Isolation of nucleic acids on surfaces

ABSTRACT

New processes and equipment to isolate and purify nucleic acids on surfaces are provided. The invention focuses on processes which use surfaces, for example, porous membranes, on which the nucleic acids are immobilized in a simple manner from the sample containing the nucleic acids and can be released again by way of simple procedural steps, whereby the simple performance of the process according to the invention makes it possible to perform the processes specifically in a fully automatic manner. An additional aspect of the present invention focuses on binding the nucleic acids to an immobile phase, especially to a membrane, in such a way and manner, that they can be released without difficulty during an additional reaction stage from this phase and, if desired, can be used in other applications, such as restriction digestion, RT, PCR or RT-PCR, or in any of the suitable analyses or enzyme reactions mentioned in the disclosure. Special isolation devices are provided that can be used to carry out the processes according to the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of pending Internationalapplication no. PCT/EP99/02664, filed Apr. 20, 1999 and designating theUnited States, and of pending International application no.PCT/EP98/06756, filed Oct. 23, 1998 and designating the United Statesand claiming priority to German application DE 19746874.8, filed Oct.23, 1997.

FIELD OF THE INVENTION

This invention concerns new processes for the isolation and purificationof nucleic acids on surfaces.

BACKGROUND OF THE INVENTION

It has been known for a long time that the genetic origin and functionalactivity of a cell can be determined and studied by examination of itsnucleic acids. Methods of analyzing nucleic acids permit direct accessto the cause of cell activity. Such methods are therefore potentiallysuperior to indirect conventional methods such as detecting metabolicproducts. For that reason a large expansion in the number of nucleicacid analyses can be expected in the future. For instance, molecularbiological analyses are already used in many areas, for example, inmedical and clinical diagnostics, in pharmacology for the developmentand evaluation of medications, in analysis of foodstuffs as well asmonitoring food manufacturing and food inspection, in the agriculturalbusiness for breeding useful plants and animals, in environmentalanalysis, and in many research areas, including, for example, paternityanalyses, tissue typing, identification of genetic diseases, genomeanalyses, molecular diagnostics, such as the identification ofinfectious diseases, transgenic research, basic research in the area ofbiology and medicine, as well as in numerous related areas.

Through RNA analysis, especially mRNA in cells, gene activity can bedetermined directly. The quantitative analysis of transcript patterns(mRNA patterns) in cells, by way of modern molecular biology methods,such as, e.g., real-time reverse transcriptase PCR (“Real-Time RT-PCR”)or gene expression chip analyses, permit for example the recognition ofdefectively expressed genes, through which many types of disorders,e.g., metabolic diseases, infections or the generation of cancer, may berecognized. Analysis of DNA from cells by way of molecular biologicalmethods, such as, e.g., PCR, RFLP, AFLP or sequencing, permits forexample the assessment of genetic defects in or the determination of theHLA type as well as of other genetic markers.

The analysis of genomic DNA and RNA is also utilized to directly provethe existence of infectious stimuli, such as viruses, bacteria, etc.

In this connection, a general difficulty exists in the fact thatbiological and/or clinical samples must be prepared in such a way thatthe nucleic acids contained therein can be utilized directly in theanalytical method in question. It is especially important that thenucleic acids be provided in good yield, that the recovered nucleicacids be of high quality, and that there be high reproducibility, inparticular where there are a greater number of samples, in which casethe analysis should be capable of being conducted automatically.

The state of the art already includes many processes for thepurification of DNA. For example, it is known how to purify plasmid DNAfor the purpose of cloning and other experimental processes. See, e.g.,the method of Birnboim, Methods in Enzymology, 100:243 (1983). In thisprocess, a cleared lysate of bacterial origin is exposed to a cesiumchloride gradient and centrifuged for a period of 4 to 24 hours. Thisstep is usually followed by the extraction and precipitation of the DNA.This process is associated with the disadvantages that it

very apparatus-intensive, and it takes a great deal of time, isexpensive to run and cannot be automated.

Other methods in which cleared lysates are used to isolate DNA are basedon ion-exchange chromatography (e.g., Colpan et al., J. Chromatog.,296:339 (1984)) and gel filtration (e.g., Moreau et al., Analyt.Biochem., 166:188 (1987)). These processes are primarily alternatives tothe cesium chloride gradients; however they require an extensive solventsupply system, and a precipitation of the DNA fractions is necessary,since these usually contain salts in high concentrations and areextremely diluted solutions.

Marko et al., Analyt. Biochem., 121:382 (1982), and Vogelstein et al.,Proc. Nat. Acad. Sci., 76:615 (1979), have found that if the DNA fromextracts containing nucleic acids is exposed to high concentrations ofsodium iodide or sodium perchlorate, only DNA will adhere to glassscintillation tubes, fiberglass membranes or fiberglass sheets that havebeen finely ground by mechanical means, while RNA and proteins will not.The DNA that has been bound in this manner can be eluted, for example,with water.

For example, in international publication WO 87/06621, theimmobilization of nucleic acids on a PVDF membrane is described.However, the nucleic acids bound to the PVDF membrane are not eluted inthe next step; instead the membrane, together with all the bound nucleicacids is introduced directly into a PCR reaction. Finally, in thisinternational patent application and in the other literature, it isstated that hydrophobic surfaces or membranes must in general be wettedbeforehand with water or alcohol, in order to be able to immobilize thenucleic acids with yields that are satisfactory.

On the other hand, for a number of modern applications, such as, forexample, the PCR, reversed transcription PCR, SunRise, LCR,branched-DNA, NASBA, or TaqMan technologies and similar real-timequantification methods for PCR, SDA, DNA and RNA chips and arrays forgene expression and mutation analyses, differential display analyses,RFLP, AFLP, cDNA synthesis or substractive hybridization, it isabsolutely necessary to be able to release the nucleic acids directlyfrom the solid phase. In this connection, WO 87/06621 teaches that,while the nucleic acids can indeed be recovered from the membranes usedin the process, this recovery is fraught with problems and is far fromsuited to the quantitative isolation of nucleic acids. In addition, thenucleic acid obtained in this manner is, comparatively, extremelydiluted, which makes subsequent isolation and concentration stepsabsolutely necessary.

SUMMARY OF THE INVENTION

According to the present invention, all aqueous or other solutions ofnucleic acids, as well as all materials and all samples containingnucleic acids, as well as biological samples and materials, foodstuffs,etc. are defined as “nucleic acid samples”. In the sense of the presentinvention, a sample or a material containing a nucleic acid is definedas a nucleic acid sample and/or a sample preparation which contains thenucleic acids in question. Biological material and/or biological samplesin this connection include, e.g., cell-free sample material, plasma,body fluids—such as for example, blood, sputum, urine, feces, sperm,cells, serum, leucocyte fractions, crusta phlogistica, smears; tissuesamples of any type, tissue parts and organs; foodstuff samples whichcontain free or bound nucleic acids or nucleic acid-containing cells;environmental samples which contain free or bound nucleic acids ornucleic acid-containing cells, plants and parts of plants, bacteria,viruses, yeasts and other fungi, other eukaryotes and prokaryotes, etc.,as they are published, e.g., in the European patent publication No. EP743 950 A1, which is incorporated herein by reference, or free nucleicacids as well. In the sense of the present invention, nucleic acidscomprise all types of nucleic acids, such as, e.g., ribonucleic acids(RNA) and desoxyribonucleic acids (DNA), in all lengths andconfigurations, such as double strands, single strand, circular andlinear, branched, etc.; monomer nucleotides, oligomers, plasmids, viraland bacterial DNA and RNA, as well as genomic or other non-genomic DNAand RNA from animal and plant cells or other eukaryotes, tRNA, mRNA inprocessed and non-processed form, hn-RNA, rRNA and cDNA as well as allother nucleic acids that can be envisioned.

For the reasons stated above, the processes known from the state of theart do not constitute—particularly with regard to automation of theprocess for obtaining nucleic acids—a suitable starting point for anisolation of nucleic acid that is as simple and quantitative as possiblefrom the point of view of process engineering. The purpose of thisinvention is therefore to overcome the disadvantages of the processesknown from the state of the art for the isolation of nucleic acids andto provide a process and method which are capable of being applied orcarried out without substantial technical expenditure.

According to the present invention, the aforementioned disadvantages aresolved by the processes, isolation and/or reaction devices uses,automatic apparatus kits according to the description, drawings andclaims below.

In addition, the invention focuses on processes which make use ofsurfaces, e.g., porous membranes, on which the nucleic acids can beeasily immobilized from the sample containing the nucleic acids, and canagain be released by way of similarly easy steps of the process, wherebythe simple performance of the process according to the invention makesit possible to specifically carry out the process in a fully automatedmanner.

Another purpose of this invention is, in particular, to bind nucleicacids to an immobile solid phase—especially to a membrane—in such amanner that in a subsequent reaction step they can be releasedimmediately from this phase and, if desired, used in other applications,such as, for example, restriction digest, RT, PCR or RT-PCR, as well asany other suitable analytical or enzymatic reaction named above.

Within the scope of the present invention, a surface is defined as anymicroporous separating layer. This may also directly rest on asubstratum and therefore only be accessible from one side or be standingfreely in space. Within the meaning of the present invention a membraneis defined as a separating layer which is accessible from both sideswhen it does not rest with its entire surface area on an impenetrablesubstratum but is entirely free or is only supported at single points.

Within the meaning of the present invention, isolation is defined as anyaccumulation of nucleic acids, in which the concentration of nucleicacids is increased and/or the portion of non-nucleic acids in a samplepreparation and/or sample is reduced.

The invention provides a process to isolate nucleic acids including thefollowing steps:

-   -   applying at least one nucleic acid sample to a membrane;    -   immobilizing the nucleic acids on the membrane;    -   releasing the immobilized nucleic acids from the membrane; and    -   removing the released nucleic acids through the membrane,        whereby the membrane contains nylon, polysulfone,        polyethersulfone, polycarbonate, polyacrylate, acrylic        copolymer, polyurethane, polyamide, polyvinylchloride,        polyfluorocarbonate, polytetrafluoroethylene, polyvinylidene        fluoride, polyethylene-tetrafluoroethylene-copolymerisate,        polybenzimidazole,        polyethylene-chlorotrifluoroethylene-copolymerisate, polyimide,        polyphenylene sulfide, cellulose, cellulose-mix-ester,        cellulose-nitrate, cellulose-acetate, polyacrylnitrile,        polyacrylnitrile-copolymers, nitrocellulose, polypropylene        and/or polyester.

Other membranes also, such as those mentioned below in the presentdescription, may be used for processes according to the invention.

Preferably the loading process takes place from the top and the removalprocess is carried out in a downward direction; however, flow-throughprocesses, for example, can be envisioned in which a horizontal columnis loaded from one side with a solution containing nucleic acid, which,after immobilization of the nucleic acids, penetrates through themembrane and can be removed at the other end of the column.

Preferably, the membrane is situated in a container, e.g., the columnmentioned above or any elongated container having an inlet and anoutlet, wherein the membrane stretches across the entire diameter of thecontainer.

The membrane may be coated so as to render it hydrophobic orhydrophilic.

Isolation processes to date, especially in isolation columns, functionwith relatively thick membranes and/or fleeces in order to achieve acomplete isolation of the nucleic acids. When the solution is suctionedthrough the membrane, however, a relatively large, so-calleddead-space-volume, i.e., the volume of the membrane, is generated fromwhich the nucleic acids can only be recovered by way of a largerquantity of an elution buffer. This, however, causes the nucleic acidsto be more diluted after the elution, which is undesirable ordisadvantageous for many applications. For this reason, a preferredembodiment of the invention uses a membrane which is less than 1 mmthick, preferably less than 0.5 mm, and most preferably less than 0.2mm, e.g., 0.1 mm thick.

The invention furthermore involves a process to isolate nucleic acidswith the following steps:

-   -   applying at least one nucleic acid sample to a surface;    -   immobilizing the nucleic acids on the surface; and    -   releasing the immobilized nucleic acids from the surface with an        elution agent.

This process is characterized in that the release takes place at atemperature whose upper limit is 10° C. or less and whose lower limit isat the freezing point of the elution agent to be used for such release,so that the elution agent does not freeze. Therefore the followinginequation applies: 10° C.≧T≧T_(S, EM), in which T is the releasetemperature and T_(S, EM) is the freezing point of the elution agent. Wehave discovered that, contrary to widespread opinion, a release of thenucleic acids near the freezing point of the elution agent is quitepossible. Such an elution at low temperature even has the unexpectedadvantage that the nucleic acids are treated more gently and that theactivity from any nucleases (DNases or RNases) still present in thesample drops practically to nothing near the freezing point, so thatdegradation of the nucleic acids is reduced or completely prevented.

Accordingly, the temperature during elution should preferably be evenlower, e.g., at less than 5° C. The lower limit may also be at 0° C. or−5° C., if the specimen is still liquid at this temperature, based onits ion content. The upper temperature limit should if possible also below, e.g., at about 5° C.

The process according to the invention therefore requires cooling of theelution buffer and may require cooling of any additional solutions used,as well as cooling of the isolation device if necessary. Since coolingcannot always be guaranteed in a reliable manner, especially duringexaminations performed in the field, e.g., when screening human samplesin developing countries, the present invention also provides anisolation device which allows isolation of nucleic acids at lowtemperatures independent from any external cooling. For such situations,the instant invention provides an isolation device to isolate nucleicacids having at least an upper part with a top opening, a bottom openingand a membrane, which is located at the bottom opening and which fillsthe entire diameter of the upper part; a bottom part with an absorbentmaterial; and a collar surrounding the upper part, at least in the areaof the membrane, which contains a coolant. The collar containing thecoolant allows cooling of the membrane and the solutions placed on themembrane such as the lysate, washing buffer and elution buffer at lowtemperatures, so that the final elution can take place in a reliablemanner within the desired temperature range near the freezing point ofthe elution buffer.

In an embodiment of this isolation device, the collar has twocompartments, which are separated from one another by a mechanicallydestructible or frangible separation wall, with each of the compartmentscontaining a solution and in which upon mixing of both solutions afterdestruction of the separating wall, the coolant is generated. Theseparating wall can be destroyed by the user, e.g., by pressing againstthe external collar wall, e.g., at points provided for such purpose, andthus causing the separating wall to tear. Suitable solutions to fill thecompartments are familiar to practitioners in the area of chemicalcooling technology. These may be adjusted to the desired temperaturesand to the outside temperatures expected when using the isolationdevice.

When recovering nucleic acids from biological samples, such as thesamples indicated above, it is often necessary to make a lysate thecells or secretions first, in order to be able to reach the nucleicacid. The lysates thus produced may also contain large amounts ofundesirable substances in addition to the nucleic acids, such asproteins or fats. If the content of such substances in a lysate is toohigh, the membrane may become clogged when the lysate is applied, whichreduces the efficiency of the nucleic acid isolation and which reducesthe permeability of the membrane during washing or elution. In order toavoid this undesirable effect, the invention provides a process in whichundesirable substances are removed before they reach the membrane.

In preferred embodiments, the process according to the invention toisolate nucleic acids comprises the following steps:

-   -   adjusting at least one nucleic acid sample to binding conditions        which allow immobilization of the nucleic acids contained in at        least one of the nucleic acid samples on a surface;    -   applying at least one nucleic acid sample to the surface; and    -   immobilization of the nucleic acids on the surface,        characterized in that before and/or after adjusting the binding        conditions, a pretreatment is applied.

The pre-treatment may, for instance, take place by salting out or byfiltration, centrifugation, enzymatic treatment, temperature effect,precipitation and/or extraction of the nucleic acid solution and/orbinding contaminants of the nucleic acid solution to surfaces. Thepre-treatment may also involve mechanical disruption or homogenizing thenucleic acid solution, if it is for example the lysate of a biologicalsample.

The binding conditions that were adjusted may permit the immobilizationof RNA and/or DNA in this case.

A pre-treatment may be necessary especially in cases when one intends toisolate biological samples with severe contaminants. The biologicalsample may consist of any conceivable material which is used eitherimmediately or can be recovered from another biological sample. Forinstance, this may be blood, sputum, urine, feces, sperm, cells, serum,leukocyte fractions, crusta phlogistica, smears, tissue samples, plants,bacteria, fungi, viruses and yeasts, as well as all other types ofbiological samples mentioned above.

The process according to the invention may be used to its greatestadvantage if the biological sample contains a large amount ofundesirable substances.

After immobilization of the nucleic acids from the pre-treated nucleicacid sample, the usual isolation steps can be followed, i.e.:

-   -   releasing the immobilized nucleic acids from the surface;    -   recovering the nucleic acids released from the surface.

A special advantage of the isolation process according to the inventionconcerns the fact that it may be connected with chemical reactions, towhich the nucleic acids are subjected directly on the surface. A varietyof analytical techniques for nucleic acids may therefore be used withthe nucleic acids isolated on the surface. In this case it is possibleto again release the nucleic acids from the surface prior to thereaction in order to guarantee their free accessibility. Alternatively,a suitable reaction may also be performed with the nucleic acids whichare directly bound on the surface.

Accordingly, one aspect of the invention involves a process with apre-treatment, as outlined above, which is characterized in that thefollowing step preferably takes place at least once after the releasestage:

-   -   performing at least one chemical reaction with the nucleic        acids.

A special advantage of this process lies in the fact that prior to thechemical reaction, no loss resulting from transfer of the nucleic acidsfrom the isolation device to a reaction device occurs, because theisolation and chemical reaction can take place in the same device.

In an additional aspect not related to pre-treatment, the inventioninvolves a process to carry out a nucleic acid amplification reactionwith the following steps:

-   -   applying at least one nucleic acid sample to a surface;    -   immobilizing the nucleic acids on the surface; and    -   performing an amplification reaction with the nucleic acids.

Especially with the small quantities of material commonly used inamplification reactions or available for use in amplification reactions,it is generally advantageous if the whole reaction sample of nucleicacids can be used in the reaction without any loss from transfer. Thisis especially advantageous for an automated process since all steps canbe carried out in one device. Furthermore, the amount of waste isreduced and the process is faster and more cost-effective.

The amplification reaction may be an isothermal or a non-isothermalreaction. The amplification reaction may, e.g., consist of anSDA-reaction (“strand displacement amplification”), a PCR, RT-PCR, LCRor a TMA or a rolling circle amplification.

A NASBA-reaction is also possible with this process according to theinvention.

Prior to carrying out the amplification reaction, the nucleic acids maybe released from the surface with a reaction buffer, whereby the eluateis located on or in the membrane. Alternatively, the amplificationreaction may be carried out in a reaction buffer that does not produce arelease of the nucleic acids from the surface.

This process preferably produces these additional steps:

-   -   if necessary, release of the reaction products from the surface        (to the extent these were still bound during the reaction); and    -   removal of the released reaction products from the surface.

Another aspect involves a process to perform chemical reactions withnucleic acids by way of the following steps:

-   -   applying at least one nucleic acid sample to a surface;    -   immobilizing nucleic acids on the surface;    -   releasing the immobilized nucleic acids from the surface;    -   performing at least one chemical reaction with the nucleic        acids; and    -   removal of the nucleic acids from the surface without prior        immobilization.

In this process the nucleic acids are no longer bound (immobilized) tothe membrane after the chemical reaction, but removed without binding.Although the elimination of such an additional step may compromise thepurity of the removed specimen, it may be preferred because it savestime in critical applications and it also simplifies certain applicationmethods. A wide range of chemical reactions is available as a result ofthe process according to the invention. Within the meaning of theinvention “chemical reaction” should be defined in this connection asany interaction of the nucleic acids with other substances (with theexception of the surface, since this “reaction” occurs in all processesdescribed herein), i.e., enzymatic modifications, hybridization withprobes, chemical sequencing reactions, pH-value-changes, e.g., for basicdepurination of RNA and acid depurination of DNA, as well as antibodybinding and protein binding. Generally, each reaction, whether itconcerns the changing of covalent bonds or hydrogen bonds, is included.

One advantage of the process according to the invention is thepermanent, spatial combination of a volume chamber, in which a greatvariety of processes can take place, and a membrane to which nucleicacids can be bound. Simply put, this combination allows the manipulationof nucleic acids followed by binding to a membrane. This is especiallyadvantageous for automated processes. After binding to the membrane, thenucleic acids are available for additional treatment steps, e.g., asmentioned above, for isolation of highly pure nucleic acids or forperforming chemical reactions with the nucleic acids. An additionalaspect of the invention makes it also possible to immediately subjectthe nucleic acids still bound to the membrane to further analysis, inorder to determine certain properties of the nucleic acids.

For that reason the invention also involves a process to analyze nucleicacids in an isolation device with the following steps:

-   -   making available an isolation device with a membrane located        therein;    -   applying at least one nucleic acid sample to the isolation        device;    -   immobilizing the nucleic acids on the membrane;    -   leading the fluid components of the sample through the membrane;        and    -   analyzing at least one property of the nucleic acids on the        membrane located in the isolation device.

After passing the fluid components through the membrane, at least onechemical reaction as mentioned above can be performed with the nucleicacids in an additional embodiment. This may serve, e.g., to allow thesubsequent analysis of the nucleic acids. Examples of reactions in thiscontext are the hybridization of probes, the radioactive labeling ofnucleic acids bound to the membrane or the binding of specificantibodies. Auxiliary reactions such as staining nucleic acids, e.g.,with intercalating substances such as ethidium bromide should also beconsidered as a chemical reaction.

Various properties of nucleic acids are open to an analysis while theyare bound to the membrane. They have already been described forconventional membranes without a combined reaction device. Some of theproperties that can be analyzed are the radioactivity of nucleic acidsor their binding affinity for molecules, in which the molecules forexample may be antibodies or dye molecules that bind nucleic acids orare bound to nucleic acids or proteins.

This process represents a considerable simplification of the analysis ofnucleic acids, since the manipulation of the free membrane is no longernecessary. This is now located in the isolation device.

An irreversible bond of the nucleic acids to the membrane, e.g., forsubsequent analytical steps is also within the scope of the presentinvention. This long-lasting or irreversible bond permits themanipulation of the membrane and the nucleic acids bound thereon to anextent that is not possible for reversibly bound nucleic acids.

An additional aspect of the invention focuses on the quantitativeprecipitation of nucleic acids.

In previously known methods based on anion-exchange chromatography forpurification of 100 μg and more plasmid-DNA (hereinafter indicated as“large scale” DNA purification), the plasmid-DNA is eluted in a highsaline buffer from the column during the last step. In order to separatethe plasmid-DNA from the salt on the one hand, and to concentrate it onthe other, it is precipitated with the aid of alcohol (e.g.,isopropanol) and centrifuged in a suitable device. The centrifugationpellet thus obtained is washed with 70% ethanol, in order to remove theresidual traces of salt and is then again subjected to centrifugation.The pellet from the second centrifugation is typically dissolved in asmall amount of low saline buffer and the plasmid-DNA is processedfurther in this form.

In addition, the state of the art has proposed processes in which DNA isadded in such a form by adding chaotropic salts to the high salinebuffer so as to cause binding to silica membranes. After a correspondingwashing step, the DNA can again be released from the membrane by way ofa low saline buffer.

A similar application is described in a publication (Ruppert et al.,Analytical Biochemistry, 230: 130-134 (1995)) in which on a small scale(isolation of less than 100 μg of plasmid-DNA) DNA precipitated withisopropanol is bound to PVDF-membranes with pore sizes of less than 0.2μm, subsequently washed with ethanol and then eluted with TE(Tris-EDTA). However, there is no description of such a method for thelarge scale process.

The DNA precipitation described with subsequent centrifugation isextremely time-consuming (approx. 1 hour), and furthermore requires theuse of centrifuges. In addition to the time factor for this procedure,the last step described for plasmid preparation is particularly prone toerrors. A partial or complete loss of the DNA-pellet also occursoccasionally. A decisive roll appears to be the type (material) of thecentrifugation device used.

The use of chaotropic salts (also described) and the subsequent bindingof nucleic acids to silica membranes is also time-consuming; moreover,because of the introduction of chaotropic salts to the preparation thereis the risk of contamination of the finally isolated DNA.

The filtration of alcoholic precipitates on a small scale as describedabove has the disadvantage that the operation cannot be transferredlinearly to a large scale process. Conventional membranes only permitthe isolation of small amounts of nucleic acids, as the membranes arequickly saturated with nucleic acids and no longer absorb anything. Whenthe precipitate buffer is removed and washed, a large portion of thenucleic acids is frequently lost again. In order to avoid this loss, theinvention also involves a process to precipitate nucleic acids by way ofthe following steps:

-   -   making available an isolation device with at least one membrane        situated therein;    -   applying a nucleic acid sample to the isolation device;    -   precipitation of the nucleic acids contained in the sample with        alcohol, so that the nucleic acids are at least bound to a        membrane. The process is characterized in that the pore size of        at least one membrane is the same or greater than 0.2        micrometers.

Alcohols considered to perform the process according to the inventionare first of all hydroxyl derivates of aliphatic or acyclical saturatedor unsaturated hydrocarbons.

Among the aforementioned hydroxyl compounds, the C₁-C₅ alkanols, such asmethanol, ethanol, n-propanol, n-butanol, tert.-butanol, n-pentanol ormixtures thereof are preferred. Especially preferred is the use ofisopropanol to carry out the process according to the invention.

In this process, the alcohol can be mixed with this solution before orafter loading the isolation device with the solution containing thenucleic acid. The volume ratio of the nucleic acid-containing solutionto alcohol, especially isopropanol, preferably is 2:1 to 1:1, mostpreferably 1.67:1 to 1:1, and for example 1.43:1.

The surface of the membrane is preferably chosen so that all the nucleicacids contained in the solution can be bound to the membrane.

The invention also involves the use of membranes with a pore size ofequal or larger than 0.2 μm to bind the alcohol-precipitated nucleicacids, which may consist of DNA and/or RNA. Especially advantageous isthe use of a 0.45 μm cellulose acetate or cellulose nitrate filterand/or the use of various layers of a 0.65 μm cellulose acetate orcellulose nitrate filter. The procedure can both be used as vacuumfiltration and as pressure filtration.

The process according to the invention permits a time-saving transfer ofnucleic acids from a high-salt buffer system to a low-salt buffersystem, which is possible without use of complex apparatus. It issuitable as a substitute for the classical alcoholic precipitation ofDNA from a high-salt buffer, which is typically by centrifugation steps.Because of the great effectiveness of the method (minor loss of yield)it is especially suitable as a preparation for a large scale process.Furthermore the process according to the invention does not introduceany additional substances in the already purified nucleic acids. Inaddition, compared to the classical method, susceptibility to errors isless (loss of the centrifugation sediment during the washing cycle isnot possible using the process of the invention).

Preferably, applying the solution should take place from the top in thevarious processes explained above. In principle, a wide range of methodsare available which pass various solutions such as nucleicacid-containing immobilization buffers, washing buffers, eluate, etc.through the membranes.

This may be achieved through gravity, centrifugation, vacuum, positivepressure (on the loading side), and capillary forces.

Between the immobilization and the separation step, the immobilizednucleic acids may be washed with at least one washing buffer. Thewashing preferably consists of the following steps for each washingbuffer:

-   -   applying a predetermined quantity of washing buffer to the        surface, and    -   passing the washing buffer through the surface.

The application and immobilization of the nucleic acids may againconsist of the following steps:

-   -   mixing of the nucleic acid sample with an immobilization buffer;    -   applying the nucleic acid sample with the immobilization buffer        on the surface, and    -   passing the liquid components through the surface in essentially        the direction of the loading step.

The processes have the major advantage that they can easily beautomated, so that at least one of the steps can be fully automated inan automatic device. It is also possible to have all steps of theprocesses performed in a pre-arranged sequence by an automaticapparatus. Especially in these cases, but also for manual handling, itis possible that a majority of nucleic acids are simultaneously subjectto isolation. For example, multi-isolation devices may be used in theform of commonly available “multi-well” devices with 8, 12, 24, 48, 96or more single isolation wells.

The removal of the nucleic acids may take place in two roughly differentdirections. On the one hand it is possible to feed (pass) the (eluted)nucleic acids that were removed through the membrane and to remove themtoward the membrane's side, that is located opposite the side on whichthe nucleic acid-containing solution and/or the lysate was placed. Inthis case the nucleic acid is removed in the direction of its passingthrough the membrane. The other possibility consists of removing thenucleic acids from the membrane and/or from the surface on the sidewhere they were introduced. The removal then takes place in thedirection opposite to their introduction or “in the same direction”, inwhich they were introduced; in other words, on the side where they wereintroduced. In this case the nucleic acids do not pass through themembrane. In some of the processes according to the invention, removalof the nucleic acids takes place through the membrane in the directionthey were introduced. In the event a process is carried out with asurface that does not have a non-permeable substratum, e.g., a syntheticlayer, the removal can of course only take place in the direction ofintroduction (hence in the opposite direction). For a few processes,however, the substance can be removed in both directions.

If the nucleic acids are eluted (released) from the surface essentiallyin the opposite direction from the direction in which they wereintroduced and immobilized, “the same direction” is essentiallyconsidered each direction with an angle equal or smaller than 180°,compared to the direction of introduction, so that upon elution, thenucleic acids under no circumstances permeate the surface, e.g., amembrane, but are removed from the surface in the direction oppositefrom the loading direction in which they were introduced to the surface.In preferred embodiments, on the other hand, the other buffers, i.e.,those buffers which contain nucleic acids during the loading process,and if required a washing buffer, are suctioned through the surface orotherwise transferred. If the isolation takes place on a membranelocated in a device, whereby the membrane fills the entire diameter ofthe device, the preferred loading method is from the top. In this casethe removal step again occurs upward. FIG. 2 shows an example of afunnel-shaped isolation device, which is loaded from the top and inwhich the removal of the nucleic acids takes place in an upwarddirection.

It is understood that, in the case of removal in a direction opposite tointroduction, other configurations are also imaginable, e.g., removal ofthe nucleic acids from below. It is possible, for example, to suction abuffer containing nucleic acids, such as a lysate buffer from a reactiondevice directly into an isolation device by way of a suctioninstallation, so that the nucleic acids will be bound to the bottom of amembrane in the isolation device. In such a case, the removal of thenucleic acids from the surface can be carried out, in such a way that anelution buffer is suctioned up from below and is drained again downwardinto a device after separation of the nucleic acids. The removal of thenucleic acids therefore also takes place in a downward direction.

A lateral removal of the nucleic acids is also possible, e.g., if ahorizontal column with a membrane located therein is loaded with alysate during the flow-through process and the horizontal column issubsequently washed with elution buffers on the side of the membrane towhich the nucleic acids are bound.

An example for the maximum possible angle of 180° is a slope with asurface suitable to bind nucleic acids along which surface the varioussolutions and/or buffers flow. Like all buffers, the elution buffer alsoarrives from one side and is drained on the other side. In this case,the inflow direction of the buffer and the draining direction of thebuffer with the nucleic acids included therein make an angle of 180°;the removal, however, continues to take place on the same side of thesurface as the immobilization.

Following the process according to the invention, the sample containingnucleic acids described above is added to a solution which contains theappropriate salts and/or alcohol(s); subsequently the sample is lysed,if necessary, and the mixture obtained in this manner is led by way of avacuum, centrifugation, positive pressure, capillary forces or by way ofother appropriate processes, through a porous surface, whereby thenucleic acids are immobilized on the surface.

Suitable salts for the immobilization of nucleic acids on membranes orother surfaces and/or for the lysis of nucleic acid samples are salts ofmetal cations, such as alkaline or alkaline earth metals, with mineralacids; especially alkaline or alkaline-earth halides and/or sulfates orphosphates, including the halides of sodium, lithium or potassium ormagnesium sulfate, which are most preferable. Other metal cations, e.g.,Mn, Cu, Cs or Al, or the ammonium cation can be used, preferably assalts of mineral acids.

Furthermore to carry out the process according to the invention, saltshaving one or more basic functions or even polyfunctional organic acidswith alkaline or alkaline-earth metals are suitable. These especiallyinclude sodium, potassium or magnesium salts with organic dicarboxylicacids, such as e.g., oxalic, malonic or succinic acids, or with hydroxyand/or polyhydroxycarboxylic acids, such as, e.g., with citric acids,preferably.

The substances indicated above to immobilize the nucleic acids onsurfaces and/or for the lysis of nucleic acid samples may be usedseparately or in mixtures, if this should prove to be more suitable forcertain applications.

In this connection the use of so-called chaotropic agents has proved tobe particularly effective. Chaotropic substances are able to disrupt thethree-dimensional structure of hydrogen bonds. This also weakens theintramolecular binding forces which are involved in the formation ofspatial structures, such as, e.g., primary, secondary, tertiary orquaternary structures, in biological molecules. Suitable chaotropicagents are well known to those skilled in the art (see, Römpp, Lexikonder Biotechnologie, Publisher H. Dellweg, R. D. Schmid and W. E. Fromm,Thieme Verlag, Stuttgart 1992).

According to this invention preferred chaotropic substances are saltsfrom the group of trichloroacetates, thiocyanates, perchlorates, iodidesor guanidinium hydrochloride and urea. The chaotropic substances arethen used in a 0.01 to 10 molar aqueous solution, preferably in a 0.1 to7 molar aqueous solution, and most preferably in a 0.2 to 5 molaraqueous solution. In this connection the aforementioned chaotropicagents can be used individually or in combination. Most preferably 0.01to 10 molar aqueous solutions, or 0.1 to 7 molar aqueous solutions, or0.2 to 5 molar aqueous solutions of sodium perchlorate, guanidiniumhydrochloride, guanidinium isothiocyanate, sodium iodide and/orpotassium iodide are used.

The salt solutions used in the processes according to the invention forlysis, binding, washing and/or for elution are preferably buffered. Allsuitable buffer systems can be considered as buffer substances, such as,e.g., carboxylic acid buffers, especially citrate buffers, acetatebuffers, succinate buffers, malonate buffers as well as glycine buffers,morpholino-propane-sulfone-acids (MOPS) or Tris (hydroxymethyl)aminomethane (Tris) in concentrations of 0.001 to 3 mol/liter,preferably 0.005 to 1 mol/liter, and most preferably 0.01 to 0.5mol/liter, and particularly preferred 0.01 to 0.2 mol/liter.

To carry out the process according to the invention, first all hydroxylderivates of aliphatic or acyclical saturated or unsaturatedhydrocarbons are eligible as alcohols. It is irrelevant whether thesecompounds contain one, two, three or more hydroxyl groups—such aspolyvalent C₁-C₅ alkanols, e.g., ethylene glycol, propylene glycol orglycerin.

In addition, the alcohols that can be used according to the inventionalso include sugar derivates, the so-called aldites, as well as phenols,e.g., polyphenols.

Among the aforementioned hydroxy compounds, C₁-C₅-alkanols, such asmethanol, ethanol, n-propanol, tert.-butanol and pentanols, or mixturesof such alcohols, are most preferred.

Within the meaning of this invention, such substances and/or membraneswhich by their chemical nature easily mix with water or absorb water areconsidered hydrophilic.

Within the meaning of this invention, such substances and/or membraneswhich by their chemical nature do not penetrate water or vice-versa andwhich cannot stay dissolved in water are considered hydrophobic.

Within the meaning of this invention, any microporous separating layeris understood to be a surface. In the case of a membrane the surfaceconsists of a film made of polymer material.

The polymer preferably consists of monomers with polar groups.

In a further embodiment of the process according to the invention, theconcept of surface furthermore also comprises a layer of particlesand/or a granulate as well as fibers such as silica gel fleece.

When hydrophobic membranes are used in the practice of this invention,membranes are preferred which consist of a hydrophilic basic materialand which are made hydrophobic by a corresponding chemicalpost-treatment which is known from the state of the art. Membranes suchas commercially available hydrophobic nylon membranes are preferablyused.

According to the invention membranes that are hydrophobic are generallydefined as those membranes which are originally hydrophilic membranesthat have been coated with hydrophobical coating agents mentioned below.Such hydrophobical coating agents coat the hydrophilic substances with athin film of hydrophobic groups, which, e.g., include longer alkylchains or siloxane groups. Many suitable hydrophobic coating agents areknown and include, e.g., paraffins, waxes, metallic soaps, etc., ifnecessary with additions of aluminum, zirconium salts, quaternaryorganic compounds, ureic derivates, lipid-modified melamine resins,silicones, zinc organic compounds, glutaric dialdehydes, and similarcompounds.

According to the invention suitable hydrophobic membranes also are thosemembranes which are by themselves hydrophobic or which have been madehydrophobic and whose basic material may contain polar groups. Accordingto these criteria, e.g., especially hydrophobic materials from thefollowing group are suitable for use according to the invention: Nylon,polysulfones, polyether sulfones, cellulose nitrate, polypropylene,polycarbonates, polyacrylates as well as acrylic copolymers,polyurethanes, polyamides, polyvinyl-chloride, polyfluorocarbonates,polytetrafluoroethylene, polyvinylidene fluoride,polyethylene-tetra-fluoroethylene copolymerisates,polyethylene-chlorotrifluoro-ethylene-copolymerisates, or polyphenylenesulfide, as well as cellulose and cellulose-mix esters, celluloseacetate or nitrocellulose as well as polybenzimidazoles, polyimides,polyacryl nitriles, polyacrylnitril-copolymers, hydrophobisized glassfiber membranes, including hydrophobisized nylon membranes which aremost preferable.

Preferred hydrophilic surfaces include hydrophilic materials per se andalso hydrophobic materials which have been hydrophilisized. For instancethe following substances can be used: hydrophilic nylon, hydrophilicpolyether-sulfones, hydrophilic polycarbonates, hydrophilic polyesters,hydrophilic poly-tetra-fluoroethylenes on polypropylene tissues,hydrophilic polytetrafluoroethylenes on polypropylene fleece,hydrophilisized polyvinylidene fluoride, hydrophilisizedpolytetrafluoroethylenes, hydrophilic polyamides, nitrocellulose,hydrophilic polybenzimidazoles, hydrophilic polyimides, hydrophilicpolyacryl-nitriles, hydrophilic polyacrylnitril-copolymers, hydrophilicpolypropylene, cellulose nitrate, cellulose-mix-ester and celluloseacetate.

The membranes described above are already known in the art, partiallyfor their use in nucleic acid binding, but not yet in the context of theinvention. A series of materials for this particular use is, however,not known from the state of the art. The extensive trials disclosedherein have demonstrated that there are additional membranes that aresuitable to bind nucleic acids.

The present invention therefore also involves the use of celluloseacetate, non-carboxylized, hydrophobic polyvinylidene fluoride, ormassive hydrophobic poly-tetra-fluorethylene as a material on which toprecipitate and isolate nucleic acids. In this context, the term“massive” denotes a material which generally consists of thecorresponding compound and is neither coated nor applied as a coating ona carrier material.

The material may be used as a membrane, as granulate, as fibers or inother suitable forms. The fibers may, e.g., be configured as fleece andthe granulate may be pressed as a grid.

The membranes used in the process described above according to theinvention (with the exception of isopropanol precipitate) for instancehave a pore diameter of 0.001 to 50 μm, preferably 0.01 to 20 μm, andmost preferably a pore size of 0.05 to 10 μm. In case the nucleic acidsare precipitated with isopropanol according to the process describedabove, the pore size must be greater than 0.2

The salts or alcohols described above or the phenols or polyphenols mayalso be considered as washing buffers. Detergents and natural substancesin the broadest sense of the word, such as albumin, or milk powder mayalso be used for the washing steps. The addition of chaotropicsubstances is also possible. Polymers as well as detergents withdissolving abilities and similar materials may also be added. Thewashing buffers and the substances contained therein should at any rategenerally be able to bind undesirable contaminants, to dissolve them orto react with them, so that these contaminants or their decompositionproducts can be removed jointly with the washing buffer.

The temperatures during the washing stage typically range from about 10°to 30° C., preferably at room temperature, although higher or lowertemperatures may also be applied successfully. When elution is performedat a low temperature, e.g. 2° C., one should not forget to also cool thewashing buffer in order to pre-cool the temperature of the isolationdevice and the surface and/or membrane to the desired temperature. Oneapplication for low temperatures is cytoplasmatic lysis, during whichthe cell nuclei remain undamaged. Higher temperatures of the washingbuffers on the other hand cause better dissolution of the contaminantsto be washed out.

Suitable eluting agents for the purposes of the invention are water oraqueous salt solutions. Buffer solutions that are known from the stateof the art are used as salt solutions, such as morpholinopropanesulfonic acid (MOPS), tris(hydroxymethyl)aminomethane (TRIS),2-[4-(2-hydroxyethyl)piperazino]ethane sulfonic acid (HEPES) in aconcentration from 0.001 to 0.5 moles/liter, preferably 0.01 to 0.2moles/liter, most preferably 0.01 to 0.05 molar solutions. Alsopreferred for use are aqueous solutions of alkaline or alkaline-earthmetal salts, in particular, their halogenides, for example, including0.001 to 0.5 molar (preferably 0.01 to 0.2 molar, most preferably 0.01to 0.05 molar) aqueous solutions of sodium chloride, lithium chloride,potassium chloride or magnesium chloride. Also preferred for use aresolutions of salts of the alkaline or alkaline-earth metals withcarboxylic or dicarboxylic acids, e.g., oxalic acid or acetic acid, orsolutions of sodium acetate or sodium oxalate in water, e.g., in theconcentration range mentioned above, such as 0.001 to 0.5 molar,preferably 0.01 to 0.2 molar, most preferably from 0.01 to 0.05 molar.

The addition of subsidiary compounds such as detergents or DMSO is alsopossible. If a chemical reaction must be carried out with the elutednucleic acids, either directly on the membrane or in another reactiondevice, it is also possible to add such substances or other subsidiarycompounds which are to be used in the reaction to the elution buffer.For instance, the addition of DMSO in low concentrations is customary inmany reactions.

After a chemical reaction with the nucleic acids, these can also beeluted with the reaction buffer. For instance, the nucleic acids can beeluted with the reaction buffer or the reaction master mix after a SDA-or a NASBA-reaction.

Most specifically, pure water is the preferred elution agent, e.g.,demineralized, bi-distilled, or ultra pure millipore water.

The elution can, for example, be carried out successfully attemperatures from below 0° C. to 90° C., e.g., from 10° to 30° C. or athigher temperatures. It is also possible to elute with water vapor. Thelower limit of the elution temperature is, as explained above, thefreezing point of the elution buffer.

Based on the smooth executability of the processes according to theinvention which can also be performed “in the field”, i.e., outside ofestablished laboratory installations and therefore without extensiveelectrically powered equipment, the invention also involves thepreparation of isolation devices with which the process according to theinvention can be carried out with a minimum of additional subsidiarymaterials. For this, a reaction device can be used which contains amembrane. This can be brought into contact with an absorbent material,such as a sponge, in order to absorb the various buffers used throughthe membrane. The sponge acts therefore as a combination vacuum pump orcentrifuge in conjunction with a waste collector. In order to recoverthe eluate, contact of the absorbent material with the membrane iseliminated, so that the eluate cannot be lost, but instead can beremoved or studied further.

In this aspect, the invention specifically involves an isolation deviceto isolate nucleic acids with at least a cylindrical upper part with atop opening, a bottom opening and a membrane which is located at thebottom opening and fills the entire diameter of the upper part; isequipped with a bottom part containing an absorbent material; and amechanism for the connection between the upper and lower parts, inwhich, after the connection has been made, the membrane is in contactwith the absorbent material, and when the connection is not made, themembrane is not in contact with the absorbent material.

Preferably, the bottom or lower part is a cylinder with the samediameter as the upper part. In this manner, a simple tube is obtainedhaving essentially a constant diameter, which can be handled in the sameway as traditional reaction devices. Especially if the upper part or theupper part plus lower part create a tube which can be placed in reactiondevice holders, such as those used in laboratories, this effect can beachieved. The mechanism can be a connection which allows a spatialseparation of the upper and lower parts, for example a bayonet socket, aplug-in socket or a threaded end. A bayonet socket has the advantagethat it is easier to lock and unlock, whereas the threaded connectionallows for a better, more watertight connection of the upper and lowerparts. Alternatively, a pre-determined breaking point can be providedbetween the upper and lower parts, which at least allows for theone-time separation of both parts and which can be manufactured at avery low price. Alternatively, the connection can also be a slidingmechanism which can be slid between the absorbent material and themembrane. In this embodiment, a separation of membrane and absorbentmaterial can be achieved as well.

To increase the processing capacity and to be able to carry out theprocess according to the invention even more economically, it is alsopossible to modify the isolation device according to the inventiondescribed above in such a way that various upper parts are placed on abottom part. The bottom part can serve simultaneously as a holder of theassembly and in addition have such dimensions that a variety ofisolation processes, at least more than mere connections for the upperand lower parts, are available, and can be carried out before thesuction capacity of the absorbent material in the bottom part isexhausted.

The absorbent material in the lower part may contain a sponge and/or agranulate. The granulate can consist of a superabsorbent material, as isknown by those skilled in the art of absorption technology (e.g., forhygiene-related items).

The invention similarly involves utilization of this isolation deviceaccording to the invention for the analysis of properties of nucleicacids and to isolate nucleic acids.

With respect to the separate stages, the processes according to theinvention are typically carried out as follows:

When starting from biological samples, they must first be subjected tolysis in the appropriate buffers. Additional processes to achieve lysismay be needed, e.g., a mechanical action, such as homogenization orultrasound, enzymatic reaction, temperature changes or additives. Incase it is required or desirable, a pre-treatment can follow this lysisin order to remove debris from the lysate. Subsequently, in case thishas not happened yet, the conditions in the lysate are adjusted, so thatimmobilization of the nucleic acids on the surface can take place. Evenafter adjustment of the binding conditions, a pre-treatment step canfollow cumulatively or alternatively to the above pre-treatment step.

This pretreated lysate of the sample used for the recovery of nucleicacids or the originally free nucleic acid(s)—if one did not start from abiological sample—is/are pipetted, for example, in a (plastic) column,in which the hydrophobic membrane is fastened, for example, on thefloor. It is more efficient if the membrane is fastened to a grid, whichserves as a mechanical support. The lysate is then conducted through themembrane, which can be achieved by applying a vacuum at the outlet ofthe column. The transport can also be accomplished by applying positivepressure to the lysate. In addition, as mentioned above, the transportof the lysate can take place by centrifugation or by the effect ofcapillary forces. The latter can be produced, for example, with asponge-like material which is introduced below the membrane and is incontact with the lysate or filtrate. In the case of centrifugation, theisolation device open at the bottom may be used in a collection tube forthe flowthrough liquid.

The washing stage included in the preferred embodiments can take placeif the washing buffer is transported through the surface of the membraneor is remaining on the same side of the surface as the nucleic acids. Ifthe washing buffer is transported or suctioned through, this can takeplace in different ways, e.g., by a sponge located on the other side ofthe membrane, a suction or positive pressure mechanism or bycentrifugation or gravity.

The advantage of a configuration utilizing an absorbent, possibly spongymaterial is that it provides a simple, secure and handy means fordisposing of the filtrate, in this case only the sponge, which by thattime is more or less saturated with the filtrate and needs to bereplaced. At this point it is clear that the column can be operatedcontinuously or also in a batch-like manner, and that both modes ofoperation can be fully automated, until the membrane is saturated withnucleic acids. In the last stage, if required, the elution of thenucleic acids takes place, which for example can be pipetted or liftedfrom the membrane or can be removed upward in another way, if no in situanalysis of the nucleic acids that are still bound is to be performed.

The desired nucleic acids are present in very small volumes of bufferswith no or low salt to concentrations, which is a great advantage forall molecular biological analyses, since it is always desirable to havepure nucleic acids in high concentrations and in the smallest volumespossible. In order to obtain the smallest possible volumes of eluate, itis especially preferred to use as surfaces those membranes that are asthin as possible, so that only very little liquid can accumulate inthem.

Furthermore, the present invention offers the advantage that in the caseof a vertical configuration of the device (where the membrane is placedin a horizontal direction) the volume located above the membrane can beused as a reaction chamber. Hence, it is possible, for example, afterisolation and removal of the nucleic acids recovered according to theprocess of the invention, to not remove them immediately but to leavethem in the isolation device and to subject them to a molecularbiological application, such as restriction digest, RT, PCR, RT-PCR, invitro transcription, NASBA, rolling circle, LCR (ligase chain reaction),SDA (strand displacement amplification) or enzyme reactions, such asRNase- and DNase-digestion for the complete removal of any of thenucleic acids that are not wanted, to bind the nucleic acids resultingfrom these reactions again to the membrane according to the processaccording to the invention or to leave them in the supernatant, ifnecessary to wash them as described, and subsequently to elute them, toisolate and/or analyze them, e.g., by way of chromatography,spectroscopy, fluorometry, electrophoresis, or similar measurements.

The nucleic acids isolated according to the invention are free ofenzymes that degrade the nucleic acids and have such a high purity thatthey can immediately be used and processed in the greatest variety ofways.

The nucleic acids produced according to the invention can be used forcloning and as substrates for a great variety of enzymes, such as, e.g.,DNA-polymerases, RNA-polymerases such as, e.g., T7-polymerase orT3-polymerase, DNA-restriction enzymes, DNA-ligase, reversetranscriptase and others.

The nucleic acids produced by the processes of the invention areespecially suitable for amplification, especially for PCR, stranddisplacement amplification, rolling circle processes, ligase chainreaction (LCR), SunRise, NASBA and similar processes.

The processes according to the invention are furthermore extremelysuitable to produce nucleic acids for their use in diagnostics, e.g., infood analysis, in toxicological examinations, in medical and clinicaldiagnostics, in diagnostics of germs, gene expression analysis, and inenvironmental analysis. The processes are especially suitable for adiagnostic process, which is characterized in that the nucleic acidspurified by way of the processes according to the invention areamplified in a subsequent step, and the nucleic acids that are thusamplified are detected subsequently and/or simultaneously (see, e.g.,Holland et al., 1991, Proc. Natl. Acad. Sci., 88: 7276-7280; Livak etal., 1995, PCR Methods Applic., 4: 357-362; Kievits et al., 1991, J.Virol. Meth., 35: 273-286; Uyttendaele et al., 1994, J. Appl.Bacteriol., 77: 694-701).

Moreover, the processes according to the invention are especiallysuitable to produce nucleic acids which, in a subsequent step, aresubjected to a signal amplification step based on a hybridizationreaction, which is specifically characterized by the fact that thenucleic acids produced in the process according to the invention arebrought into contact with “branched nucleic acids”, especially branchedDNA and/or branched RNA and/or corresponding dendritic nucleic acids andthe signal that is generated is detected, as described in the followingliterature (e.g., Bresters et al., 1994, J. Med. Virol., 43(3): 262-286;Collins et al., 1997, Nucl. Acids Res., 25(15): 2979-2984).

An example of automation of a process according to the invention isexplained below and examples to perform the process with differentsurfaces and nucleic acids are also described. In this descriptionreference is made to the attached figures which illustrate thefollowing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows automatic equipment suitable to perform the processaccording to the invention in a stylized graph.

FIG. 2 shows a first embodiment of an isolation device and collector toperform the process according to the invention.

FIG. 3 shows a second embodiment of an isolation device and collector toperform the process according to the invention.

FIG. 4 shows a third embodiment of an isolation device and collector toperform the process according to the invention.

FIG. 5 shows embodiments of isolation devices with an upper partaccording to the invention.

FIG. 6 shows the ethidium bromide stained gel of an electrophoreticseparation of various samples according to the process of the invention.

FIG. 7 shows another ethidium bromide stained gel of an electrophoreticseparation of various samples according to the process of the invention.

FIG. 8 shows another ethidium bromide stained gel of an electrophoreticseparation of various samples according to the process of the invention.

FIG. 9 shows the ethidium bromide stained gel of an electrophoreticseparation of various samples according to the process of the invention.

FIG. 10 shows another ethidium bromide stained gel of an electrophoreticseparation of various samples according to the process of the invention.

FIG. 11 shows another ethidium bromide stained gel of an electrophoreticseparation of various samples according to the process of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The processes according to the invention are preferably performed in anautomatic manner either partially or completely, in other words, in allstages. An example for suitable automatic equipment is illustrated inFIG. 1, in which a main part 1 is equipped with control electronics anddriving engines with a work platform 3 and a movable arm 2. Variouselements are positioned on the work platform, such as area 4 to holdvarious devices. A vacuum manifold 5 serves to absorb liquids fromisolation devices which are placed above it and are open at the bottom,or otherwise with the devices connected to the vacuum manifold. A shaker6 is also provided, which can be used, e.g., for the lysis of biologicalsamples. The isolation device assemblies used are, e.g.,injection-molded parts with integrated isolation devices, in which thesurfaces according to the invention are included. Typically 8, 12, 24,48, 96 or up to 1536 isolation devices can be used as these areavailable for example in the formats of modern multi-well-plates. Evenhigher numbers of isolation devices might be possible in one plate, ifstandards are available. With the aid of Luer-adapters it is, however,also possible to make separate bottoms of the assembly available and toequip these with one or more isolation devices as needed. Isolationdevices used individually without Luer-adapters are also included in theinvention.

Under a vacuum and dispensing mechanism 8 the isolation devices areplaced in the automatic apparatus and with these, liquids can be takenup and dispensed. In this assembly several single vacuum units may beprovided, so as to make the simultaneous processing of an isolation orreaction device possible. The vacuum and dispensing mechanism 8therefore acts as a pipet. Vacuum and pressure are fed to the vacuum anddispensing mechanism 8 via tube 9.

To isolate the nucleic acids, reaction devices with cells may forexample be placed in the shaker/holder 6, into which lysis buffers areintroduced with the help of the dispensing mechanism. After mixing, thecell lysates are transferred to isolation devices. The lysis buffer issubsequently passed through the surfaces in the isolation devices.Subsequently, the surfaces may be washed with a washing buffer in orderto remove cell lysate residues, in which also the washing buffer isdrained off downward. Finally, an elution buffer is dispensed into theisolation devices and after repeated shaking the separated nucleic acidsare removed from above and transferred to collection microtubes.

Usually, disposable tips are used on the vacuum and dispensing mechanism8 to prevent contamination of the samples.

FIGS. 2 through 4 show different schematic examples for suitableisolation devices to be used according to the present invention.

In FIG. 2, a funnel-shaped isolation device 10 is provided with asurface 11, e.g., a membrane, which is placed on a collector 12, whichcontains a sponge-like material 13 that serves to absorb the lysis andwashing buffers. Under the sponge-like material 13 a superabsorbentlayer 14 may be placed to improve the suction performance.Alternatively, layer 14 may also contain a material which is chemicallyable to react with water, e.g., acrylate. The water is therefore alsoremoved from the process. Lysate or another preparation of nucleic acidsis placed in the funnel. The sponge-like material 13 absorbs the appliedliquid through membrane 11. Prior to the addition of the elution buffer,the sponge is moved some distance from the membrane, e.g., by amechanism inside a collector 12 (not visible in the drawing). This willprevent the elution buffer in the last stage from being also suctionedthrough membrane 11. This buffer, however, stays on the surface (FIG. 2b) and can be removed together with the nucleic acids from above. Whenusing this assembly, the vacuum mechanism 5 in the automatic apparatusis no longer necessary.

FIG. 3 shows another example of an isolation device, which is connectedto a collector 16 via a Luer-connection located at the bottom via aLuer-adapter 17, which in this case does not contain a sponge, but isconnected to a vacuum mechanism via a muff 18. Lysis and washing buffersmay in this case be suctioned through membrane 11 by creating a vacuum(FIG. 3 a). When the eluate buffer is introduced, the vacuum remainsturned off, so that the eluate can be removed from above (FIG. 3 b).With the use of a Luer-connection, individual isolation devices can beremoved from the isolation device assembly. It will be understood,however, that the vacuum collector can also be combined with fixedisolation devices, e.g., multi-well devices containing 8, 12, 24, 48, 96or more single devices.

FIG. 4 finally shows an embodiment which provides a collector, intowhich the buffers are suctioned through the membrane or surface 11 byway of gravity or centrifuged. The eluate buffer, which is used in smallvolumes, is not heavy enough itself to penetrate membrane 11 and canagain be removed from above (FIG. 4 b).

FIG. 5 shows embodiments of the isolation devices according to theinvention.

In FIG. 5A, an isolation device with a cylindrical upper part 20 hasbeen illustrated. This upper part is connected to a bottom part 22 byway of a threaded connection 25. Instead of the threaded connectionother types of connections may also be used, to the extent these permita watertight connection of the upper and bottom parts and provide apossibility of introducing membrane 11. In this embodiment, membrane 11is applied directly to the bottom opening of upper part 20. It may,however, also be moved inward or be placed at an angle other than 90°with respect to the upper part's wall. The bottom part also has acylindrical shape, but may be of a different design in otherembodiments. For example, a quadrangular shape may be used, whichimproves the stability of the upper part 20 on a surface. The wideningof bottom part 22 compared to upper part 20 is also possible, forexample in case a larger cavity is required in bottom part 22 in certainembodiments of the process according to the invention in order to fullyabsorb the solutions used in the absorbent material 13.

An alternative embodiment to the embodiment shown in FIG. 5A isillustrated in FIG. 5B. In this case upper part 20 and bottom part 22are fixed to one another or may also be built in one piece. Between theabsorbing material 13 and membrane 11, a sliding mechanism 27 may beslid via an opening 26 into the isolation device to separate membrane 11and absorbent material 13 from one another. In this example slidingmechanism 27 is equipped with an additional handle 28, which facilitatespulling out sliding mechanism 27. The sliding mechanism can, however,also be designed without this handle. As shown in FIG. 5B, the absorbentmaterial 13 expands slightly, to be able to bridge the space taken up bythe sliding mechanism and to make contact with the membrane.

FIG. 5C shows another embodiment of the isolation device according tothe invention. In this case the bottom part 23 is equipped with severalconnections 30 to accommodate the upper parts 20, thus permitting thesimultaneous processing of a multiplicity of samples. The upper parts 20in this example are connected with bottom part 23 by way of threadedconnections 31. Although shown smaller in the illustration than theupper parts 20 of FIGS. 5A and 5B, it is understood that the upper partscan be the same size (or can be larger or smaller) as indicated in thoseembodiments.

Finally, FIG. 5 D shows an isolation device according to the inventionwith a collar 32 with coolant, which surrounds membrane 11 on theoutside. In this embodiment, upper part 20 and bottom part 24 areconnected to one another by way of a plug-in socket. Another type ofconnection or a one-piece version are, however, also possible. Collar 32consists of two compartments, 33 and 34, which can be connected with oneanother by destroying the separating wall 35. Both compartments 33, 34are loaded with substances, e.g. solutions, which, when mixed afterdestruction of the separating wall 35, causes the temperature of theentire mixture to drop.

The invention described above will be further explained in the followingexamples. Different and alternative designs of the devices and processeswill become clear to the skilled practitioner from the description aboveand from the following examples. It should expressly be pointed out,however, that these examples and the description accompanying theseexamples only serve as an illustration of the invention and are not tobe considered a limitation of the invention.

EXAMPLE 1 Isolation of Total RNA from HeLa Cells

Commercially available nylon membranes (for example, a material fromMSI, “Magna SH” with a pore diameter of 1.2 μm, or a material from PallGmbH, “Hydrolon” with a pore diameter of 1.2 μm), which are chemicallypost-treated and to be hydrophobic, were placed as a single layer in aplastic column. The membranes were placed on a polypropylene grid whichserved as a mechanical support. The membranes were fixed in the plasticcolumn with a ring. The column prepared in this manner was connected bymeans of a Luer connection to a vacuum chamber. All the isolation stepswere carried out through the application of a vacuum.

For the isolation, 5×10⁵ HeLa cells were harvested by centrifugation andthe supernatant removed. The cells were lysed by the addition of 150 μlof a commercial guanidium isothiocyanate buffer (e.g., RLT buffer fromQIAGEN GmbH, Hilden, Del.), in a manner thoroughly familiar to thoseskilled in the art. Lysis was promoted by roughly mixing by pipetting orvortexing for 5 seconds. Then 150 μl of 70% ethanol were added and mixedin by repeatedly pipetting or by vortexing for about 5 seconds.

The lysate was transferred into the plastic column and suctioned throughthe membrane by evacuating the vacuum chamber. Under these conditions,the RNA remained bound to the membrane. Next, washing was performedusing a first commercial washing buffer containing guanidiumisothiocyanate (e.g., with RW1 buffer from QIAGEN GmbH) and, after that,with a second washing buffer containing TRIS or TRIS and alcohol (e.g.,with the RPE buffer from QIAGEN GmbH). The washing buffers in each casewere suctioned through the membrane by evacuation of the vacuum chamber.After the final washing step, the vacuum was maintained for a period ofabout 10 minutes, in order to dry the membrane, after which the vacuumwas switched off.

For the elution, 70 μl RNase-free water was pipetted onto the membranein order to dissolve the purified RNA from the membrane. Afterincubation for one minute at a temperature in the range from 10° to 30°C., the eluate was pipetted from the membrane from above and the elutionstep was repeated in order to make sure that the elution was complete.

The quantity of isolated total RNA obtained in this manner wasdetermined by spectrophotometric measurement of the light absorption at260 nm. The ratio between the absorbance values at 260 and 280 nm givesan estimate of RNA purity.

The results of the two isolations with hydrophobic nylon membranes (Nos.1 and 2) are shown in Table 1, compared with experiments in which on theone hand a hydrophilic nylon membrane (Nyaflo) (No. 3) and a silicamembrane (No. 4) were used. The values reported in the table provideconvincing support for the impressive isolation yield and separationeffect of the materials used in accordance with this invention. Theyalso show that silica gel-fleece clearly produces a lower yield, whichpresumably can be attributed to its fleecelike structure and the ensuingabsorption of a large portion of the eluate buffer.

TABLE 1 RNA yield and purity of total RNA isolated according toExample 1. Yield of Total-RNA Absorbance Sample No. Type of Membrane(μg) E₂₆₀/E₂₈₀ 1 Magna SH 1.2 μm 6.0 1.97 (hydrophobic nylon) 2 Hydrolon1.2 μm 7.1 2.05 (hydrophobic nylon) 3 Nylaflo (hydrophilic <0.2 Notnylon) Determined 4 hydrophilic silica <0.2 Not membrane Determined

The isolated RNA can also be analyzed on agarose gels that have beenstained with ethidium bromide. For this purpose, for example, 1.2%formaldehyde agarose gels were prepared. The result is shown in FIG. 6.In FIG. 6, Lane 1 is the total RNA that was isolated on a hydrophobicnylon membrane (Magna SH, Sample no. 1) with a pore diameter of 1.2 μm.Lane 2 is total RNA that was isolated by means of a hydrophobic nylonmembrane (Hydrolon, Sample no. 2) with a pore diameter of 1.2 μm. Lane 3represents the chromatogram of a total RNA that was isolated by means ofa silica membrane (Sample no. 4). In each case, 50 μl of the total RNAeluate was analyzed. FIG. 6 provides convincing evidence that when asilica membrane was used, no measurable proportion of the total RNA canbe isolated.

EXAMPLE 2 Isolation of Free RNA by Binding the RNA to HydrophobicMembranes by Means of Various Salt-Alcohol Mixtures

In this example, the lysate and washing solutions are conducted throughthe hydrophobic membrane by applying a vacuum.

Hydrophobic nylon membranes (e.g., 1.2 μm Hydrolon from Pall) wereintroduced into plastic columns connected to a vacuum chamber, in amanner similar to that of Example 1. To 100 μl aliquots of an aqueoussolution containing total RNA were added 350 μl of a commerciallyavailable lysis buffer containing guanidium isothiocyanate (e.g., RLTbuffer from QIAGEN), 350 μl of 1.2 M sodium acetate solution, or 350 μlof 4 M lithium chloride solution, respectively, and the resultingsolutions were mixed by pipetting.

Next, 250 μl of ethanol were added to each mixture and mixed, likewiseby pipetting. After that, the solutions containing RNA were transferredinto the plastic columns and suctioned through the membrane byevacuating the vacuum chamber. Under the conditions described, the RNAremains bound to the membranes. The membranes were then washed, asdescribed in Example 1. Finally, the RNA, also as described in Example1, was removed from the membrane by pipetting from above.

The quantity of isolated total RNA was determined by spectrophotometricmeasurement of the light absorption at 260 nm. The ratio between theabsorbance values at 260 and 280 nm gives an estimate of RNA purity. Theresults are set forth in Table 2 below.

TABLE 2 Isolation of RNA from aqueous solution by binding the RNA tohydrophobic membranes using various salt-alcohol mixtures. Yield ofTotal Sample RNA Absorbance No. Salt/Alcohol mixture (μg) E₂₆₀/E₂₈₀ 1RLT-Buffer QIAGEN/35% Ethanol 9.5 1.92 2 0.6 M Sodium Acetate/35%Ethanol 8.5 1.98 3 1 M Sodium Chloride/35% Ethanol 7.9 1.90 4 2 MLithium Chloride/35% Ethanol 4.0 2.01

EXAMPLE 3 Isolation of Total RNA from HeLa Cells

Following the procedures of Example 1, plastic columns were assembledwith different hydrophobic membranes. Each column thus prepared wasplaced in a collection tube, and the following isolation steps wereperformed by way of centrifugation.

For the isolation, 5×10⁵ HeLa cells were harvested by centrifugation andthe supernatant removed. The cells were lysed by the addition of 150 μlof a commercially available guanidinium isothiocyanate buffer, such as,e.g., RLT-buffer from QIAGEN, using well known procedures. In thisconnection, lysis is encouraged by multiple pipetting or by vortexingfor 5 seconds. Subsequently, 150 μl of 70% ethanol was added and mixedby multiple pipetting or by vortexing for 5 seconds.

The lysate was subsequently transferred into a plastic column and passedthrough the membrane by centrifugation at 10000×g for 1 minute.Subsequently, washing was performed with a commercially availablewashing buffer containing guanidinium isothiocyanate, e.g., with theRW1-buffer of QIAGEN, followed by a second washing step using a buffercontaining TRIS and alcohol, e.g., RPE-buffer from QIAGEN. The washingbuffers were passed through the membrane by centrifugation. The lastwashing step takes place at 20000×g for 2 minutes to dry the membrane.

For elution, 70 μl of RNase-free water were pipetted onto the membraneto release the purified RNA from the membrane. After a 1-2 minuteincubation at a temperature between 10°-30° C., the eluate was takenfrom above by pipetting from the membrane. The elution step was repeatedonce to achieve complete elution.

The quantity of isolated total RNA was determined by spectrophotometricmeasurement of the light absorption at a wavelength of 260 nm. RNAquality is determined by spectrophotometric determination of the lightabsorption ratio compared at 260 nm and at 280 nm. The isolation resultswith different hydrophobic membranes are listed in Table 3 below. Thedata represent the average of 3-5 parallel tests per membrane. Using asilica membrane, no measurable quantity of total RNA could be isolated,where the eluate was recovered by removing it from above from themembrane.

MSI Magna-SH, 5 μm Hydrophobic Nylon 10.2 1.71 MSI Magna-SH, 10 μmHydrophobic Nylon 7.36 1.76 MSI Magna-SH, 20 μm Hydrophobic Nylon 7.041.65 Sartorius Type 118 Hydrophobic Poly- 7.6 1.61 TetrafluoroethyleneMupor PM12A Hydrophobic Poly- 6.7 1.77 Tetrafluoroethylene Mupor PM3VLHydrophobic Poly- 6.6 1.77 Tetrafluoroethylene

EXAMPLE 3b Isolation of Total RNA from HeLa-Cells by Binding toHydrophilic Membranes

Using the procedures of Example 1, plastic columns were assembled usingdifferent hydrophilic membranes. Each column thus prepared was placed ina collection tube, and the following isolation steps were performed bycentrifugation.

For the isolation, 5×10⁵ HeLa cells were used. The isolation steps andelution of the nucleic acids were carried out as described above inExample 3 for hydrophobic membrane columns.

The quantity of isolated total RNA was determined by spectrophotometricmeasurement of the light absorption at a wavelength of 260 nm. RNAquality was determined by the spectrophotometric determination of theratio of the light absorption compared at 260 nm and at 280 nm. Theisolation results with various hydrophilic membranes are listed in Table3b below. The data represent the average of 2-5 parallel tests permembrane. Using a silica membrane, no measurable quantity of total RNAcould be isolated, where the eluate was recovered by removing it fromabove from the membrane.

TABLE 3b Yield of total RNA isolated by binding to hydrophilicmembranes. Manufacturer Membrane Material RNA (μg) E₂₆₀/E₂₈₀ PallLoprodyne Hydrophilic Nylon 3.1 1.8 Pall Loprodyne Hydrophilic Nylon 3.11.78 Pall Biodyne A Hydrophilic Nylon 3.1 1.8 Pall Biodyne A HydrophilicNylon 3.6 1.83 Pall Biodyne B Hydrophilic Nylon 2.6 1.84 Pall Biodyne BHydrophilic Nylon 4.2 1.84 Pall Biodyne C Hydrophilic Nylon 6.1 1.88Pall Biodyne C Hydrophilic Nylon 5.2 1.91 Pall Biodyne plus HydrophilicNylon 3.3 1.87 Pall I.C.E.-450 Hydrophilic Polyethersulfone 6.36 1.8Pall I.C.E.-450sup Hydrophilic Polyethersulfone 3.07 1.71 Pall Supor -800 Hydrophilic Polyethersulfone 4.12 1.7 Pall Supor - 450 HydrophilicPolyethersulfone 4.69 1.69 Pall Supor - 100 Hydrophilic Polyethersulfone3.25 1.71 Pall Hemasep V Hydrophilic Polyester 4.16 1.74 Pall Hemasep LHydrophilic Polyester 6.67 1.65 Pall Leukosorb Hydrophilic Polyester 1.51.84 Pall Premium Release Hydrophilic Polyester 1.66 1.63 Membrane PallPolypro -450 Hydrophilic Polypropylene 5.09 1.78 Gore-Tex OH 9339Hydrophilic Poly- 1.08 1.65 Tetrafluoroethylene Gore-Tex OH 9338Hydrophilic Poly- 3.97 1.67 Tetrafluoroethylene Gore-Tex QH 9318Hydrophilic Poly- 3.61 1.69 Tetrafluoroethylene Millipore DuraporePolyvinylidene Fluoride made 5.6 1.69 Hydrophilic Millipore DuraporePolvinylidene Fluoride made 3.12 1.68 Hydrophilic Millipore LCRPoly-Tetrafluoroethylene 3.14 1.66 made Hydrophilic Sartorius Type 250Hydrophilic Polyamide 4.3 1.66 Sartorius Type 113 Hydrophilic CelluloseNitrate 1.8 1.86 Sartorius Type 113 Hydrophilic Cellulose Nitrate 1.91.74 Infiltec Polycone, 0.01 Hydrophilic Polycarbonate 0.17 1.64Infiltec Polycone, 0.1 Hydrophilic Polycarbonate 0.73 1.68 InfiltecPolycone, 1 Hydrophilic Polycarbonate 3.33 1.86

EXAMPLE 4 Isolation of Free RNA from an Aqueous Solution

Using the procedures according to Example 1, plastic columns wereassembled with different hydrophobic membranes. 100 μl of an aqueoussolution containing total RNA were mixed with 350 μl of a commerciallyavailable lysis buffer containing guanidinium-isothiocyanate, e.g.,RLT-buffer from QIAGEN. Subsequently, 250 μl of ethanol were added andmixed by pipetting. This mixture was then introduced to the column andpassed through by centrifugation (10000×g; 1 minute) through themembrane. The membranes were subsequently washed twice with a washingbuffer, e.g., RPE from QIAGEN. The buffer was passed through themembranes by centrifugation. The last washing step was carried out at20000×g to dry the membranes.

Next, the RNA, as described in Example 1, was eluted with RNase-freewater and removed from the membrane from above by pipetting. Thequantity of isolated total RNA was determined by spectrophotometricmeasurement of light absorption at a wavelength of 260 nm. RNA qualitywas determined by the spectrophotometric determination of the ratio ofthe light absorption at 260 nm to 280 nm. The isolation results withvarious hydrophobic membranes are listed in Table 4 below. The datarepresent the average of 3-5 parallel tests per membrane. Using a silicamembrane, no measurable quantity of total RNA could be isolated, wherethe eluate was recovered by removing it from above from the membrane.

TABLE 4 Isolation of free RNA from an aqueous solution by binding tohydrophobic membranes. Manufacturer Membrane Material RNA (μg) E₂₆₀/E₂₈₀Pall Hydrolon, 1.2 μm Hydrophobic Nylon 5.15 1.75 Pall Hydrolon, 3 μmHydrophobic Nylon 0.22 1.79 Pall Fluoro Trans G HydrophobicPolyvinylidene Fluoride 5.83 1.79 Pall Fluoro Trans W HydrophobicPolyvinylidene Fluoride 5.4 1.84 Pall Bio Trace HydrophobicPolyvinylidene Fluoride 4.0 1.79 Pall Emflon HydrophobicPoly-Tetrafluor-Ethylene 0.2 1.7 Pall Supor-450 PR HydrophobicPolyethersulfone 5.97 1.71 Pall Supor-200 PR HydrophobicPolyethersulfone 2.83 1.66 Pall V-800 R Hydrophobic Acrylatecopolymer2.74 1.77 Gore-Tex OH 9335 Hydrophobic Poly-Tetrafluor-Ethylene 4.351.63 Gore-Tex OH 9336 Hydrophobic Poly-Tetrafluor-Ethylene 7.43 1.71Gore-Tex OH 9337 Hydrophobic Poly-Tetrafluor-Ethylene 5.96 1.62 Gore-TexQH 9316 Hydrophobic Poly-Tetrafluor-Ethylene 5.92 1.67 Gore-Tex QH 9317Hydrophobic Poly-Tetrafluor-Ethylene 8.7 1.66 Millipore FluoroporeHydrophobic Poly-Tetrafluor-Ethylene 8.46 1.70 Millipore Durapore, 0.65μm Hydrophobic Polyvinylidene Fluoride 4.23 1.8 MSI Magna-SH, 1.2 μmHydrophobic Nylon 1.82 1.76 MSI Magna-SH, 5 μm Hydrophobic Nylon 0.61.78 Sartorius Type 118 Hydrophobic Poly-Tetrafluor-Ethylene 0.9 1.82Sartorius Type 118 Hydrophobic Poly-Tetrafluor-Ethylene 5.4 1.74 MuporPM12A Hydrophobic Poly-Tetrafluor-Ethylene 1.1 1.98

EXAMPLE 4b Isolation of Free RNA from an Aqueous Solution by HydrophilicMembranes

Following the procedures of Example 1, plastic columns were assembledusing different hydrophilic membranes.

100 μl of an aqueous solution containing total RNA were mixed with 350μl of a commercially available lysis buffer containingguanidinium-isothiocyanate, e.g., RLT-buffer from QIAGEN. Subsequently250 μl of ethanol were added and mixed by pipetting back and forth. Thismixture was then introduced to the column, passed through the membrane,washed and dried according to the procedure used in Example 4, above.

Finally, the RNA, as described in Example 1, was eluted with RNase-freewater and removed from the membrane using a pipette.

The quantity of isolated total RNA was determined by spectrophotometricmeasurement of the light absorption at a wavelength of 260 nm. RNAquality was determined by the spectrophotometric determination of theratio of the light absorption compared at 260 nm and at 280 nm. Theisolation results with various hydrophilic membranes are listed in Table4b below. The data represent the average from 2-5 parallel tests permembrane. Using a silica membrane, no measurable quantity of total RNAcould be isolated, where the eluate was recovered by removing it fromabove from the membrane.

TABLE 4B Isolation of free RNA from an aqueous solution by binding tohydrophilic membranes. Manufacturer Membrane Material RNA (μg) E₂₆₀/E₂₈₀Pall Loprodyne Hydrophilic Nylon 2 1.8 Pall Loprodyne Hydrophilic Nylon1.4 1.87 Pall Biodyne A Hydrophilic Nylon 4.5 1.93 Pall Biodyne AHydrophilic Nylon 3.1 1.9 Pall Biodyne B Hydrophilic Nylon 1.7 1.94 PallBiodyne B Hydrophilic Nylon 1.2 1.94 Pall Biodyne C Hydrophilic Nylon3.7 1.93 Pall Biodyne C Hydrophilic Nylon 3.1 1.93 Pall Biodyne plusHydrophilic Nylon 1.1 1.87 Pall I.C.E.-450 Hydrophilic Polyethersulfone1.92 1.82 Pall I.C.E.- Hydrophilic Polyethersulfone 0.87 1.67 450supPall Supor-800 Hydrophilic Polyethersulfone 3.93 1.74 Pall Supor-450Hydrophilic Polyethersulfone 1.78 1.74 Pall Supor-100 HydrophilicPolyethersulfone 1.04 1.68 Pall Hemasep V Hydrophilic Polyester 4 1.79Pall Hemasep L Hydrophilic Polyester 0.47 2.1 Pall Polypro- HydrophilicPolypropylene 5.09 1.78 450 Gore-Tex OH 9339 HydrophilicPoly-Tetrafluor- 0.43 1.48 Ethylene Gore-Tex OH 9338 HydrophilicPoly-Tetrafluor- 3.63 1.64 Ethylene Gore-Tex QH 9318 HydrophilicPoly-Tetrafluor- 5.92 1.67 Ethylene Millipore Durapore PolyvinylideneFluoride made 1.18 1.79 Hydrophilic Millipore LCRPoly-Tetrafluor-Ethylene made 2.84 1.72 Hydrophilic Sartorius Type 250Hydrophilic Polyamide 2.7 1.7 Sartorius Type 111 Hydrophilic CelluloseAcetate 1.6 1.85 Sartorius Type 111 Hydrophilic Cellulose Acetate 2.22.1 Sartorius Type 111 Hydrophilic Cellulose Acetate 0.3 2.01 SartoriusType 113 Hydrophilic Cellulose Nitrate 4 1.88 Sartorius Type 113Hydrophilic Cellulose Nitrate 3.8 1.87

EXAMPLE 5 Isolation of Total RNA from HeLa-Cells Depending on the PoreSize of the Membranes

Following the procedures of Example 1, plastic columns were assembledwith different hydrophobic membranes with different pore sizes.

As in Example 3, a cell lysate was made from 5×10⁵HeLa cells andtransferred to the columns. Subsequently the membranes were washed withthe commercially available buffers RW1 and RPE from QIAGEN. The lastcentrifugation step was carried out at 20000×g for 2 minutes to dry themembrane. The elution was carried out as described in Example 1.

The results are listed in Table 5 below. 3-5 parallel tests per membranewere performed and the average value calculated for each.

TABLE 5 Yield of isolated total RNA using hydrophobic membranes withdifferent pore sizes. Pore Size RNA Manufacturer Membrane Material (μm)(μg) E₂₆₀/E₂₈₀ Infiltec Polycon 0.01 Hydrophilic Polycarbonate 0.01 0.171.64 Pall Fluoro Trans G Hydrophobic 0.2 6.16 1.72 PolyvinylideneFluoride Pall Supor-450 PR Hydrophobic 0.45 3.96 1.76 PolyethersulfoneMillipore Durapore Hydrophobic 0.65 7.45 1.72 Polyvinylidene FluorideMSI Magna-SH Hydrophobic Nylon 1.2 4.92 1.69 MSI Magna-SH HydrophobicNylon 5 10.2 1.71 MSI Magna-SH Hydrophobic Nylon 10 7.36 1.76 MSIMagna-SH Hydrophobic Nylon 20 7.04 1.65

EXAMPLE 6 Stability and Quality of Isolated Total RNA from HeLa Cells

According to procedures of Example 1, plastic columns were assembledwith a commercially available membrane (Pall, Hydrolon with a 3 μm poresize).

According to the procedures of Example 3, a cell lysate was made from5×10⁵HeLa cells and transferred to the columns. Subsequently, themembranes were washed with the commercially available buffers RW1 andRPE from QIAGEN. The last centrifugation step was carried out at 20000×gfor 2 minutes to dry the membrane. The elution was carried out asdescribed in Example 1.

The isolated total RNA was left to incubate for 16 hours at 37° C. andsubsequently placed on a denaturating agarose gel and analyzed. It wasdemonstrated that the RNA did not suffer degradation. The RNA isolatedwith the method described above shows no contaminants with enzymes thatdegrade nucleic acids and therefore is of high quality.

EXAMPLE 7 Isolation of Free RNA from an Aqueous Solution by Binding to aHydrophilic Membrane in a 96-Well Plate

A 96-well plate with a hydrophilic Polyvinylidene Fluoride membrane(Durapore, 0.65 μm by Millipore) was used. 5.3 ml of an aqueous solutioncontaining total RNA were mixed with 18.4 ml of a commercially availablelysis buffer containing guanidinium isothiocyanate, e.g., RLT bufferfrom QIAGEN. Subsequently 13.1 ml ethanol were added and mixed bypipetting back and forth. For each well, 350 μl of this mixture wereintroduced and passed through the membrane by applying a vacuum. Themembranes were subsequently washed twice with a buffer, e.g., RPE fromQIAGEN. The buffer was passed through the membrane each time by applyinga vacuum. After the last washing step, the plate was dabbed once with apaper towel and subsequently dried for 5 minutes by applying a vacuum.

The RNA was eluted as described in Example 1, with RNase-free water andremoved from the membrane by way of a pipette. The quantity of isolatedtotal RNA was determined by spectrophotometric measurement of the lightabsorption at a wavelength of 260 nm and the average value as well asthe standard deviation for the entire plate was calculated. The averagevalue is 8.4 μg with a standard deviation of 0.7 μg.

EXAMPLE 8 Isolation of Total RNA by Way of Capillary Forces

A 96-well plate with a hydrophilic Polyvinylidene Fluoride membrane(Durapore, 0.65 μm by Millipore) was used. 33 μl of an aqueous solutioncontaining total RNA were mixed with 110 μl of a commercially availablelysis buffer containing guanidinium isothiocyanate, e.g., RLT bufferfrom QIAGEN. Subsequently 78 μl ethanol were added and mixed bypipetting. 45 μl of this mixture were introduced into each well. Anabsorbent household sponge was moistened with water, and the 96-wellplate was placed with the membrane's bottom side on the sponge. The RNAmixture was passed through the membrane by way of capillary forces. Themembranes were subsequently washed twice with a buffer, e.g., RPE fromQIAGEN. The wash buffer was also passed through the membrane by placingthe plate on the sponge. After the last washing step, the plate wasair-dried for 5 minutes.

The RNA, as described in Example 1, was eluted with RNase-free water andremoved from the membrane by way of a pipette.

The quantity of isolated total RNA is subsequently determined byspectrophotometric measurement of the light absorption at a wavelengthof 260 nm, and the average value as well as the standard deviation iscalculated. The average value is 5.9 μg with a standard deviation of 0.7μg.

EXAMPLE 9 Isolation of Genomic DNA from an Aqueous Solution by Way of aBuffer Containing Guanidinium Hydrochloride

According to Example 1, plastic columns were assembled with hydrophobicmembranes (e.g., Magna-SH, 5 μM by the MSI Company). Purification iscarried out with commercially available buffers from QIAGEN.

200 μl of an aqueous solution of genomic DNA from liver tissue wereintroduced in PBS buffers. 200 μl of a buffer containing guanidiniumhydrochloride, e.g. QIAGEN's AL, were added to and mixed with thissolution. Subsequently 210 μl of ethanol were added and mixed throughvortexing. The mixture was introduced to the column according to Example3 and passed through the membrane by way of centrifugation. The membranewas then washed and dried with an alcohol containing buffer, e.g.,QIAGEN's AW. The elution was performed as described in Example 1. Threeparallel tests were carried out and the average value calculated. Theamount of isolated DNA is subsequently determined by spectrophotometricmeasurement of the light absorption at a wavelength of 260 nm and isapprox. 30% of the starting amount. The absorption ratio at 260 nm to280 nm is 1.82.

EXAMPLE 10 Isolation of Genomic DNA from an Aqueous Solution by Bindingto Hydrophobic Membranes by Way of a Buffer Containing GuanidiniumIsothiocyanate

According to Example 1, plastic columns were assembled with differentmembranes. 100 μl of an aqueous solution containing total DNA were mixedwith 350 μl of a lysis buffer containing guanidinium isothiocyanate (4 MGITC, 0.1 M MgSO₄, 25 mM Na-Citrate, pH 4). Subsequently 250 μl ethanolwere added and mixed by pipetting. This mixture was then transferred tothe column and passed through the membrane by way of centrifugation(10000×g; 1 minute). The membranes were subsequently washed twice with abuffer, e.g., RPE by QIAGEN. The buffer was passed through the membranesby way of centrifugation. The last washing step was carried out at20000×g to dry the membranes.

The elution was performed as described in Example 1. Three paralleltests were carried out per membrane and the average value is calculatedeach time. The results are listed in Table 6.

TABLE 6 DNA-yield from an aqueous solution by binding to hydrophobicmembranes Manufacturer Membrane Material DNA (μg) Pall Hydrolon,Hydrophobic Nylon 1.3 1.2 μm Pall Supor-450 Hydrophobic Polyethersulfon2.2 PR Millipore Fluoropore Hydrophobic Poly-Tetrafluor- 1.1 EthyleneMillipore Durapore Hydrophobic Polyvinylidene 1.2 Fluoride

EXAMPLE 11 Isolation of Genomic DNA from Tissue

According to Example 1, plastic columns were assembled with hydrophobicmembranes (e.g., Magna-SH, 5 μm by MSI). Purification was carried outwith the commercially available buffers from QIAGEN.

180 μl of ATL-buffer were added to 10 mg of kidney tissue (mouse) andground in a mechanical homogenizer. Subsequently proteinase K (approx.0.4 mg dissolved in 20 μl of water) were added and incubated for 10minutes at 55° C. After adding 200 μl of a buffer containing guanidiniumhydrochloride, e.g., AL by QIAGEN, and after a 10-minute incubation at70° C., 200 μl of ethanol were added and mixed with this solution. Thismixture was transferred on to the column and passed through the membraneby centrifugation. The membrane was then washed with alcohol containingbuffers, e.g., AW1 and AW2 from QIAGEN, and subsequently dried by way ofcentrifugation. The elution was carried out as described in Example 1.Three parallel tests were carried out and the average value calculated.

The amount of isolated DNA, determined by spectrophotometric measurementof the light absorption at a wavelength of 260 nm, was on average 9.77μg. The absorption ratio at 260 nm to 280 nm was 1.74.

EXAMPLE 12 Isolation of Genomic DNA from Blood

According to the procedures of Example 1, plastic columns were assembledwith hydrophobic membranes (e.g., Magna-SH, 5 μm by MSD. Purificationwas carried out with the commercially available buffers from QIAGEN.

200 μl of AL buffer and 20 μl of QIAGEN protease were added to 200 μl ofblood, thoroughly mixed, and left to incubate for 10 minutes at 56° C.After adding 200 μl of ethanol, the solution was mixed, transferred ontothe column, and passed through the membrane by way of centrifugation.The membrane was then washed with alcohol containing buffers, e.g., AW1and AW2 from QIAGEN, and subsequently dried by way of centrifugation.The elution was carried out as described in Example 1.

The amount of isolated DNA, determined by spectrophotometric measurementof the light absorption at a wavelength of 260 nm, was 1.03 μg. Theabsorption ratio at 260 nm 280 nm is 1.7.

EXAMPLE 13 Isolation of Total RNA from an RNA-DNA-Mixture

Following the procedures of Example 1, plastic columns were assembledwith hydrophobic membranes (e.g., Hydrolon 1.2 μm by the Pall Company).275 μl of an aqueous solution containing total RNA and genomic DNA weremixed with 175 μl of a commercially available lysis buffer containingguanidinium isothiocyanate, e.g., the RLT buffer from QIAGEN. 250 μl ofethanol were added and mixed by pipetting. The mixture was transferredto the column and passed through the membrane, washed and driedaccording to Example 4. The flow-through from the first centrifugationstep was placed on a commercially available mini-spin column (e.g.,QIAamp Mini-Spin Column from QIAGEN) and passed through the membrane viacentrifugation. The remaining washing steps were performed as describedin Example 4.

After this, the nucleic acids were eluted with 140 μl of RNase-freewater by way of centrifugation (10000×g, 1 minute) and analyzed innon-denaturing agarose gel (see FIG. 7). The major part of the total RNAcan be separated from the genomic DNA with the use of the methoddescribed above.

FIG. 7 shows an ethidium-bromide stained gel of an electrophoreticseparation of two different eluates.

Lane 1: Isolation of total RNA by way of a hydrophobic nylon membrane.Lane 2: Isolation of genomic DNA from the flow-through by way of aQIAamp mini-spin column of the QIAGEN company.

EXAMPLE 14 Isolation of Plasmid DNA from an Aqueous Solution by Bindingto Hydrophobic and Hydrophilic Membranes

Following the procedures of Example 1, plastic columns were assembledutilizing different membranes.

100 μl of an aqueous solution (pCMVβ from Clontech) containing plasmidwere mixed with 350 μl of lysis buffer containing guanidiniumisothiocyanate (4 M GITC, 0.1 M MgSO₄, 25 mM sodium-acetate, pH 4).Subsequently, 250 μl of isopropanol were added and mixed by pipetting.This mixture was then transferred onto one of the columns and passedthrough the membrane, washed and dried according to the proceduresdescribed in Example 4. Finally the plasmid DNA, as described previouslyin Example 1, was eluted with RNase-free water and removed from themembrane by pipetting.

The amount of isolated plasmid DNA was determined by spectrophotometricmeasurement of the light absorption at a wavelength of 260 nm. Theisolation results using various membranes are listed in Table 7 below.Three parallel tests per membrane were carried out and each time theaverage value is calculated.

TABLE 7 Plasmid DNA-yield from an aqueous solution by binding tomembranes Manu- Plasmid facturer Membrane Material DNA (μg) PallHydrolon, Hydrophobic Nylon 1.9 1.2 μm Pall Fluoro Trans G HydrophobicPolyvinylidene 2.2 Fluoride Pall I.C.E.-450 Hydrophilic Polyethersulfone0.8 Pall I.C.E.-450sup Hydrophilic Polyethersulfone 1.5 Pall Supor-450PR Hydrophobic Polyethersulfone 4.7 Pall Supor-200 PR HydrophobicPolyethersulfone 4 Pall Supor-800 Hydrophilic Polyethersulfone 0.5 PallSupor-450 Hydrophilic Polyethersulfone 0.9 Pall Supor-100 HydrophilicPolyethersulfone 1 Pall V-800 R Hydrophobic Acrylic Copolymer 1.5 PallVersapore- Hydrophobic Acrylic Copolymer 0.2 1200R Pall Polypro-450Hydrophilic Polypropylene 1.4 Gore-Tex QH 9318 HydrophilicPoly-Tetrafluoro- 4.9 Ethylene Gore-Tex OH 9335 HydrophobicPoly-Tetrafluoro- 4.3 Ethylene Millipore Durapore, PolyvinylideneFluoride made 1.8 0.65 μm Hydrophobic Millipore Durapore, HydrophobicPolyvinylidene 1.7 0.65 μm Fluoride MSI Magna-SH, Hydrophobic Nylon 1.11.2 μm

EXAMPLE 15 Immobilization of Total RNA from an Aqueous Solution with theUse of Different Chaotropic Agents

Following the procedures of Example 1, plastic columns were assembledutilizing different hydrophobic membranes.

100 μl of an aqueous solution containing total RNA were mixed with 350μl of different lysis buffers, which contain guanidinium isothiocyanate(GITC) or guanidinium hydrochloride (GuHCl) in different concentrations.250 μl ethanol were added and mixed by pipetting. This mixture was thenplaced on one of the columns and passed through the membrane by way ofcentrifugation (10000×g; 1 minute). The membranes were subsequentlywashed twice with an alcohol containing buffer, e.g., RPE from QIAGEN.The buffer was passed through the membrane by centrifugation. The lastwashing step was performed at 20000×g to dry the membrane. The elutionwas carried out as described in Example 1. Two tests were carried out todetermine the average value. The results are listed in Table 8.

TABLE 8 RNA-yield from an aqueous solution by way of chaotropic agentsChaotropic Agents, Concentration in Binding Yield of Total MembraneSolution RNA (μg) Hydrolon, 1.2 μm GITC, 500 mM 2.3 Hydrolon, 1.2 μmGITC, 1 M 0.8 Hydrolon, 1.2 μm GITC, 3 M 0.9 Fluoro Trans G GITC, 500 mM0.4 Fluoro Trans G GITC, 1 M 1.25 Fluoro Trans G GITC, 3 M 0.6 Hydrolon,1.2 μm GuHCI, 500 mM 2.6 Hydrolon, 1.2 μm GuHCI, 1 M 6.7 Hydrolon, 1.2μm GuHCI, 3 M 2.9 Fluoro Trans G GuHCI, 500 mM 0.4 Fluoro Trans G GuHCI,1 M 1.25 Fluoro Trans G GuHCI, 3 M 0.6

EXAMPLE 16 Immobilization of Total RNA from an Aqueous Solution UsingAlcohols

Following the procedures of Example 1, plastic columns were assembledutilizing different hydrophobic membranes. 100 μl of an aqueous solutioncontaining total RNA are mixed with 350 μl of a lysis buffer containingguanidinium isothiocyanate (concentration 4 M). Different amounts ofethanol and isopropanol were added and filled with RNase-free water upto 700 μl and mixed. This mixture was then introduced to a column andpassed through the membrane and washed according to the procedures ofExample 4. The elution took place as in Example 1. Two tests werecarried out to determine the average yield. The results are listed inTable 9.

TABLE 9 RNA-yield from an aqueous solution with different alcohols in abinding solution Alcohol, Yield Concentration of Total Membrane inBinding Solution RNA (μg) Hydrolon, 1.2 μm Ethanol, 5% 0.7 Hydrolon, 1.2μm Ethanol, 30% 2.85 Hydrolon, 1.2 μm Ethanol, 50% 4.5 Durapore, 0.65 μmEthanol, 5% 0.4 Durapore, 0.65 μm Ethanol, 30% 1.25 Durapore, 0.65 μmEthanol, 50% 0.6 Hydrolon, 1.2 μm Isopropanol, 5% 0.35 Hydrolon, 1.2 μmIsopropanol, 30% 4.35 Hydrolon, 1.2 μm Isopropanol, 50% 3.2 Durapore,0.65 μm Isopropanol, 10% 1.35 Durapore, 0.65 μm Isopropanol, 30% 4.1Durapore, 0.65 μm Isopropanol, 50% 3.5

EXAMPLE 17 Immobilization of Total RNA from an Aqueous Solution withVarious pH-Values

Using the procedures described in Example 1, plastic columns wereassembled utilizing various hydrophobic membranes. 100 μl of an aqueoussolution containing total RNA were mixed with 350 μl of a lysis buffercontaining guanidinium isothiocyanate (concentration 4 M). The buffercontained 25 mM of sodium citrate and was adjusted to differentpH-values with HCl or NaOH. Subsequently, 250 μl of ethanol were addedand mixed. This mixture was then introduced to the column and passedthrough the membrane and washed according to the procedures of Example4. The elution took place as in Example 1. Two tests are carried out todetermine an average value. The results are listed in Table 10.

TABLE 10 RNA-yield from an aqueous solution with various pH-values in abinding solution pH of Yield of Total Membrane Binding Solution RNA (μg)Hydrolon, 1.2 μm pH 3 0.15 Hydrolon, 1.2 μm pH 9 1.6 Hydrolon, 1.2 μm pH11 0.05 Fluoro Trans G pH 1 0.45 Fluoro Trans G pH 9 2.85 Fluoro Trans GpH 11 0.25

EXAMPLE 18 Immobilization of Total RNA from an Aqueous Solution withVarious Salts

According to Example 1, plastic columns are assembled with hydrophobicmembranes. 100 μl of a total RNA containing aqueous solution were mixedwith 350 μl of a salt containing lysis buffer (NaCl, KCL, MgSO₄). 250 μlof H₂O or ethanol were then added and mixed. This mixture was thentransferred to a column and passed through the membrane, washed andeluted according to the procedures of Example 4. Two tests were carriedout to determine the average value. The results are listed in Table 11.

TABLE 11 RNA-yield from an aqueous solution with various salts in thebinding solution Yield of Total RNA Membrane Salt Concentration inBinding Solution (μg) Hydrolon, 1.2 μm NaCl, 100 mM; without ethanol 0.1Hydrolon, 1.2 μm NaCl, 1 M; without ethanol 0.15 Hydrolon, 1.2 μm NaCl,5 M; without ethanol 0.3 Hydrolon, 1.2 μm KCl, 10 mM; without ethanol0.2 Hydrolon, 1.2 μm KCl, 1 M; without ethanol 0.1 Hydrolon, 1.2 μm KCl,3 M; without ethanol 0.25 Hydrolon, 1.2 μm MgSO₄, 100 mM; withoutethanol 0.05 Hydrolon, 1.2 μm MgSO₄, 750 mM; without ethanol 0.15Hydrolon, 1.2 μm MgSO₄, 2 M; without ethanol 0.48 Hydrolon, 1.2 μm NaCl,500 mM; with ethanol 2.1 Hydrolon, 1.2 μm NaCl, 1 M; with ethanol 1.55Hydrolon, 1.2 μm NaCl, 2.5 M; with ethanol 1.35 Hydrolon, 1.2 μm KCl,500 mM; with ethanol 1.6 Hydrolon, 1.2 μm KCl, 1 M; with ethanol 2.1Hydrolon, 1.2 μm KCl, 1.5 M; with ethanol 3.5 Hydrolon, 1.2 μm MgSO₄, 10mM; with ethanol 1.9 Hydrolon, 1.2 μm MgSO₄, 100 mM; with ethanol 4.6Hydrolon, 1.2 μm MgSO₄, 500 M; with ethanol 2

EXAMPLE 19 Immobilization of Total RNA from an Aqueous Solution UsingVarious Buffer Conditions

Following the procedures of Example 1, plastic columns were assembledusing different hydrophobic membranes.

100 μl of an aqueous solution containing total RNA were mixed with 350μl of a lysis buffer containing guanidinium isothiocyanate(concentration 2.5 M). The lysis buffer was mixed with variousconcentrations of sodium citrate, pH 7, or sodium oxalate, pH 7.2.Subsequently 250 μl of ethanol were added and mixed. This mixture wasthen transferred to a column and passed through the membrane and elutedaccording to the process described in Example 4. The results are listedin Table 12. Two tests were carried out to determine the average value.

TABLE 12 RNA-yield from an aqueous solution with various bufferconcentrations in a binding solution Yield Na-Citrate/Na-Oxalate, ofTotal Membrane Conc. in Lysis Buffer RNA (μg) Hydrolon, 1.2 μmNa-Citrate, 10 mM 2.2 Hydrolon, 1.2 μm Na-Citrate, 100 mM 2.4 Hydrolon,1.2 μm Na-Citrate, 500 mM 3.55 Supor-450 PR Na-Citrate, 10 mM 1.1Supor-450 PR Na-Citrate, 100 mM 1.15 Supor-450 PR Na-Citrate, 500 mM 0.2Hydrolon, 1.2 μm Na-Oxalate, 1 mM 1.5 Hydrolon, 1.2 μm Na-Oxalate, 25 mM1.05 Hydrolon, 1.2 μm Na-Oxalate, 50 mM 0.9 Supor-450 PR Na-Oxalate, 1mM 1.9 Supor-450 PR Na-Oxalate, 25 mM 1.3 Supor-450 PR Na-Oxalate, 50 mM1.7

EXAMPLE 20 Immobilization of Total DNA from an Aqueous Solution UsingVarious Buffers

According to the procedures of Example 1, plastic columns were assembledwith hydrophobic membranes (for example Hydrolon 1.2 μm from the PallCompany).

100 μl of an aqueous solution containing total DNA were mixed with 350μl of a lysis buffer containing guanidinium isothiocyanate (4 M GITC,0.1 M MgSO₄). To this lysis buffer various buffer substances were added(concentration 25 mM) and adjusted to different pH-values. Subsequently,250 μl of ethanol were added and mixed. The mixture was then introducedto the column and passed through the membrane, washed and eluted as inExample 4.

The results are set forth in Table 13. Triple tests are carried out andaverage values determined.

TABLE 13 DNA-yield from an aqueous solution with various buffersubstances in a binding solution pH in the Yield of Buffer SubstanceLysis Buffer DNA (μg) Sodium Citrate pH 4 1.3 Sodium Citrate pH 5 0.6Sodium Citrate pH 6 1.4 Sodium Citrate pH 7 0.5 Sodium Acetate pH 4 0.9Sodium Acetate pH 5 1 Sodium Acetate pH 6 0.6 Sodium Acetate pH 7 0.5Potassium Acetate pH 4 0.6 Potassium Acetate pH 5 0.9 Potassium AcetatepH 6 1.2 Potassium Acetate pH 7 1.4 Ammonium Acetate pH 4 0.7 AmmoniumAcetate pH 5 0.3 Ammonium Acetate pH 6 5.7 Ammonium Acetate pH 7 1.5Glycine pH 4 0.5 Glycine pH 5 1.1 Glycine pH 6 1.6 Glycine pH 7 1.1Malonate pH 4 1.5 Malonate pH 5 0.3 Malonate pH 6 3.1 Malonate pH 7 1.6Succinate pH 4 2.8 Succinate pH 5 2.3 Succinate pH 6 2.5 Succinate pH 74.7

EXAMPLE 21 Immobilization of Total RNA from an Aqueous Solution UsingPhenol

According to the procedures of Example 1, plastic columns were assembledwith hydrophobic membranes (e.g., Hydrolon, 1.2 μm from the PallCompany).

An aqueous solution containing RNA was mixed with 700 μl of phenol andpassed through the membranes using centrifugation. The membranes werewashed and the RNA eluted as in Example 4. Two tests were carried outand an average value determined.

The amount of isolated RNA was subsequently determined byspectrophotometric measurement of the light absorption at a wavelengthof 260 nm and is on average 10.95 μg. The absorption ratio at 260 nm tothe one at 280 nm is 0.975.

EXAMPLE 22 Washing of Immobilized Total RNA Under Different SaltConcentrations

Following the procedures of Example 1, plastic columns were assembledwith hydrophobic membranes.

100 μl of an aqueous solution containing total RNA were mixed with 350μl of a lysis buffer containing guanidinium isothiocyanate(concentration 4 M). Subsequently, 250 μl of ethanol were added andmixed. This mixture was then transferred to the column and passedthrough the membrane and washed according to Example 4. The membraneswere then washed twice with a buffer containing various concentrationsof NaCl and 80% ethanol. The buffer was passed through the membrane bycentrifugation. The last washing step was carried out at 20000×g inorder to dry the membranes. The elution takes place according to theprocedure of Example 1. Two tests were carried out and an average valuedetermined. The results are listed in Table 14.

TABLE 14 RNA-yield from an aqueous solution with NaCl in the washingbuffer Yield of NaCl in the Total RNA Membrane Washing Buffer (μg)Hydrolon, 1.2 μm NaCl, 10 mM 1.4 Hydrolon, 1.2 μm NaCl, 50 mM 3.15Hydrolon, 1.2 μm NaCl, 100 mM 3 Durapore, 0.65 μm NaCl, 10 mM 2.7Durapore, 0.65 μm NaCl, 50 mM 2.85 Durapore, 0.65 μm NaCl, 100 mM 2.7

EXAMPLE 23 Elution of Immobilized Total RNA Under Different Salt andBuffer Conditions

According to the procedures of Example 1, plastic columns were assembledwith hydrophobic membranes.

100 μl of an aqueous solution containing total RNA were mixed with 350μl of a lysis buffer containing guanidinium isothiocyanate(concentration 4 M). Subsequently, 250 μl of ethanol were added andmixed. This mixture was then introduced to the column and passed throughthe membrane and washed according to the procedures of Example 4.

For elution, 70 μl of a NaCl-containing solution, a Tris/HCl buffer (pH7) or a sodium oxalate solution (pH 7.2) were pipetted onto themembrane, in order to elute the purified RNA from the membrane. After 1to 2 minutes of incubation at a temperature of 10° C.-30° C., the eluatewas pipetted from above from the membrane. The elution step was repeatedonce in order to achieve complete elution. Two tests were carried outand an average value determined. The results are summarized in Table 15.

TABLE 15 RNA-yield from an aqueous solution with NaCl, Tris/HCl orsodium oxalate in the elution buffer Yield NaCl or Tris in of TotalMembrane the Elution Buffer RNA (μg) Hydrolon, 1.2 μm NaCl, 1 mM 1.35Hydrolon, 1.2 μm NaCl, 50 mM 1.2 Hydrolon, 1.2 μm NaCl, 250 mM 0.45Durapore, 0.65 μm NaCl, 1 mM 0.9 Durapore, 0.65 μm NaCl, 50 mM 0.35Durapore, 0.65 μm NaCl, 500 mM 0.15 Hydrolon, 1.2 μm Tris/HCl, 1 mM 0.35Hydrolon, 1.2 μm Tris/HCl, 10 mM 0.75 Durapore, 0.65 μm Tris/HCl, 1 mM1.5 Durapore, 0.65 μm Tris/HCl, 50 mM 1 Durapore, 0.65 μm Tris/HCl, 250mM 0.1 Hydrolon, 1.2 μm Na-Oxalate, 1 mM 0.45 Hydrolon, 1.2 μmNa-Oxalate, 10 mM 0.65 Hydrolon, 1.2 μm Na-Oxalate, 50 mM 0.3 Durapore,0.65 μm Na-Oxalate, 1 mM 2 Durapore, 0.65 μm Na-Oxalate, 10 mM 0.155Durapore, 0.65 μm Na-Oxalate, 50 mM 0.15

EXAMPLE 24 Elution of the Immobilized RNA at Different Temperatures

Following the procedure of Example 1, plastic columns were assembledusing a hydrophobic membrane (e.g., Hydrolon, 3 μm from the PallCompany).

For isolation, 5×10⁵HeLa-cells were used. The following isolation stepswere carried out as described in Example 3.

For elution, 70 μl of RNase-free water of a different temperature werepipetted onto the membrane in order to elute the purified RNA from themembrane. After an incubation of 1-2 minutes at the correspondingelution temperature, the eluate was pipetted off the membrane fromabove. The elution step was repeated once in order to achieve completeelution. Triple tests were carried out and an average value determined.The results are summarized in Table 16.

TABLE 16 RNA-yield at different elution temperatures Yield of TotalMembrane Elution Temperature RNA (μg) Hydrolon, 3 μm Ice cold 2.2Hydrolon, 3 μm 40° C. 3.2 Hydrolon, 3 μm 50° C. 3.9 Hydrolon, 3 μm 60°C. 3.7 Hydrolon, 3 μm 70° C. 3.7 Hydrolon, 3 μm 80° C. 2.9

EXAMPLE 25 Elution of Immobilized RNA by Way of Centrifugation

Following the procedures of Example 1, plastic columns were assembledwith a hydrophobic membrane (e.g., Hydrolon 1.2 μM from the PallCompany).

100 μl of an aqueous solution containing total RNA were mixed with 350μl of a commercially available lysis buffer containing guanidiniumisothiocyanate (e.g., RLT buffer from QIAGEN). 250 μl of ethanol werethen added and mixed by pipetting. This mixture was then transferredonto the column and passed through the membrane using centrifugation(10000×g; 1 minute). The membranes were subsequently washed twice with abuffer (e.g., RPE buffer from QIAGEN). Each time the buffer was passedthrough the membranes by way of centrifugation. The last washing stepwas carried out at 20000×g in order to dry the membrane.

For elution, 70 μl of RNase-free water were pipetted onto the membranein order to elute the RNA from the membrane. After an incubation of 1minute at a temperature of 10° C.-30° C., the eluate was passed throughthe membrane by centrifugation (10000×g, 1 minute). In order to achievecomplete elution, the elution step was repeated once and the eluatesjoined together. Five parallel tests were carried out and the averagevalue calculated.

The amount of isolated total RNA was subsequently determined byspectrophotometric measurement of the light absorption at a wavelengthof 260 nm and was on average 6.4 μg. The absorption ratio at 260 nm to280 nm was 1.94.

EXAMPLE 26 Use of Total RNA in a ‘Real Time’ Quantitative RT-PCR Using5′ Nuclease PCR Assay to Amplify and Detect β-Actin mRNA

Following the procedures of Example 3, plastic columns were assembledusing a commercially available membrane (Hydrolon from Pall, with a poresize of 3 μm).

To isolate RNA, 1×10⁵ HeLa cells were used, and the purification oftotal RNA was carried out as described in Example 1. The elution wascarried out with 2×70 μl of H₂O as described in Example 1. For thecomplete removal of remaining amounts of DNA, the sample was treatedwith a DNase prior to analysis.

A “one-device ‘Real Time’ quantitative RT-PCR” was carried out with theuse of the commercially available reaction system of Perkin-Elmer(TaqMan™ PCR Reagent Kit) by using a M-MLV reverse transcriptase. Byusing a specific primer and a specific TaqMan probe for β-actin (TaqMan™β-actin Detection Kit, made by Perkin Elmer) the β-actin mRNA moleculesin the total RNA sample were first converted into β-actin cDNA andsubsequently the total reaction was amplified and detected immediately,without interruption, in the same reaction device. The reactionspecimens were produced according to the manufacturer's instructions.Three different amounts of isolated total RNA are used (1, 2, 4 μl ofeluate) and triple determination tests were carried out. As a control,three specimens without RNA were also tested.

The cDNA synthesis was carried out at 37° C. for one hour, immediatelyfollowed by a PCR which comprised 40 cycles. The reactions and theanalyses were carried out on an ABI PRISM™ 7700 Sequence Detectormanufactured by Perkin Elmer Applied Biosystems. Every amplicongenerated during a PCR-cycle produces a light emitting molecule, whichis generated by splitting from the TaqMan-probe. The total light signalthat is generated is directly proportional to the amplicon quantity thatis being generated and hence to the original amount of transcriptavailable in the total RNA sample. The emitted light is measured by theinstrument and evaluated by a computer program. The PCR cycle, duringwhich the light signal must first be detected over the background noise,will be designated as the “Threshold Cycle” (ct). This value is ameasure for the amount of specifically amplified RNA available in thesample.

For the 1 μl RNA eluate, isolated with the process described here, anaverage ct-value of 17.1 was calculated; for 2 μl in total RNA thect-value was 16.4 and for 4 μl of total RNA the ct-value was 15.3. Thisresulted in a linear dependency between the total RNA and the ct-value,indicating that the total RNA was free of substances that might inhibitthe amplification reaction. The control specimens containing no RNA didnot produce any signals.

EXAMPLE 27 Use of Total RNA in an RT-PCR for Amplification and Detectionof β-Actin mRNA

According to Example 1, plastic columns were assembled with commerciallyavailable membranes (Pall, Hydrolon with a pore size of 1.2 or 3 μm;Sartorius, Sartolon with a pore size of 0.45 μm).

For isolation of RNA, two different starting materials were used: (1)total RNA from liver (mouse) in an aqueous solution; purification,elution carried out as described in Example 4; and (2) 5×10⁵ HeLa-cells,the purification of total RNA and the elution are carried out asdescribed in Example 3.

For each test, 20 ng of isolated total RNA were used. As a control, RNAwhich was purified by way of RNeasy-Kits (QIAGEN) and a sample withoutRNA were used.

A RT-PCR was performed with these samples under standard conditions. Foramplification two different primer pairs were used for the β-actin-mRNA.A 150 bp-sized fragment serves as proof of sensitivity, a 1.7 kbp-sizedfragment assesses the integrity of the RNA. From the RT-reaction, 1 μlwas removed and introduced to the subsequent PCR. 25 cycles wereperformed for the small fragment and 27 cycles for the large fragment.The annealing temperature was 55° C. The amplified samples weresubsequently placed on a non-denaturing gel and analyzed (FIG. 8).

For the 20 ng quantity used of total RNA isolated in the processdescribed above, the corresponding DNA-fragments can be demonstrated inthe RT-PCR. When using total RNA from mouse liver, no transcript can bedemonstrated, as the conditions used here are adjusted to human β-actinmRNA. The control specimens which contain no RNA do not produce anysignals.

FIG. 8 shows ethidium bromide stained agarose gels of an electrophoreticseparation of RT-PCR reaction products.

FIG. 8A: Lanes 1 to 8: RT-PCR of the 150 by fragment:Lanes 1 & 2: RNA from mouse liver in an aqueous solution purified withthe Hydrolon 1.2 μm membrane;Lanes 3 & 4: RNA from HeLa-cells purified with the Sartolon membrane;Lanes 5 & 6: RNA from HeLa-cells purified with the Hydrolon 3 μmmembrane;Lane 7: RNA purified using the RNeasy-Mini-Kit;Lane 8: Control without RNA.FIG. 8B: Lanes 1 to 8: RT-PCR of the 1.7 kbp fragment:Lanes 1 & 2: RNA from mouse liver in an aqueous solution purified withthe Hydrolon 1.2 μm membrane;Lanes 3 & 4: RNA from HeLa-cells purified with the Sartolon membrane;Lanes 5 & 6: RNA from HeLa-cells purified with the Hydrolon 3 μmmembrane;Lane 7: RNA purified using the RNeasy-Mini-Kit;Lane 8: Control without RNA.

EXAMPLE 28 Use of Total RNA in a NASBA-Reaction (Nucleic Acid SequenceBased Amplification) for the Amplification and Detection of β-Actin mRNA

Following the procedures described in Example 1, plastic columns wereassembled with commercially available membranes (Pall, Hydrolon with apore size of 1.2 or 3 μm; Sartorius, Sartolon with a pore size of 0.45μm).

For isolation of RNA, two different starting materials were used: (1)total RNA from liver (mouse) in an aqueous solution; purification,elution carried out as described in Example 4; and (2) 5×10⁵ HeLa-cells,the purification of total RNA and the elution are carried out asdescribed in Example 3.

A NASBA-reaction is performed under standard conditions (Fahy, E. etal., 1991, PCR Methods Amplic., 1:25-33). For amplification, β-actinspecific primers were used.

For each test 20 ng of isolated total RNA are used. As a control, RNAwhich was purified by way of RNeasy-Kits (QIAGEN) and a sample withoutRNA, were used. First they were incubated for 5 minutes at 65° C. andfor 5 minutes at 41° C. Following this step, an enzyme mixtureconsisting of RNaseH, T7-polymerase and AMVV-RT was added and incubatedfor 90 minutes at 41° C. The amplified samples were subsequently placedon a non-denaturing gel and analyzed. For the 20 ng of total RNAisolated in the process described above, a specific transcript can bedemonstrated (FIG. 9).

FIG. 9 shows an ethidium-bromide stained agarose gel of anelectrophoretic separation of the NASBA-reactions.

Lanes 1 to 8: NASBA-Reactions:

Lanes 1 & 2: RNA from mouse liver purified from an aqueous solution withthe 1.2 μm Hydrolon membrane;Lane 3 & 4: RNA from HeLa-cells purified with the Sartolon membrane;Lane 5 & 6: RNA from HeLa-cells purified with the 3 gm Hydrolonmembrane;Lane 7: RNA purified using the RNeasy-Mini-Kit;Lane 8: Control without RNA.

EXAMPLE 29 NASBA-Reaction for Amplification and Detection of β-ActinmRNA on Hydrophobic Membranes

According to the procedures of Example 1, plastic columns were assembledwith commercially available membranes (Pall, Hydrolon with a pore sizeof 3 μm; Supor-450 PR with a pore size of 0.45 μm; Millipore, Fluoroporewith a pore size of 3 μm).

For the isolation of RNA, different quantities of HeLa cells were used,the purification of total RNA was carried out as described in Example 3.The elution was performed by adding 20 μl NASBA-reaction buffer. TheNASBA-reaction is subsequently performed on the membrane.

A NASBA-reaction is performed under standard conditions (Fahy, E. etal., 1991, PCR Methods Amplic., 1:25-33). For amplification, β-actinspecific primers were used.

The reaction device was first incubated for 5 minutes at 41° C. in awater bath. Following this step, an enzyme mixture consisting of RNaseH,T7-Polymerase and AMVV-RT was added and incubated for 90 minutes at 41°C. The amplified samples subsequently were placed on a non-denaturinggel and analyzed. For the quantity of RNA used and isolated from 5×10⁵to 3×10⁴ HeLa cells, a specific transcript can be observed for the totalRNA isolated by the process described here.

FIG. 10 shows an ethidium-bromide stained agarose gel of anelectrophoretic separation of the NASBA-reactions.

FIG. 10A: Lanes 1 to 4: RNA from HeLa-cells purified with the 3 μmHydrolon membrane:Lane 1: 2.5×10⁵ cells;Lane 2: 1.25×10⁵ cells;Lane 3: 6×10⁴ cells;Lane 4: 3×10⁴ cells.FIG. 10B: Lanes 1 to 3: RNA purified from HeLa-cells:Lane 1: RNA from 2.5×10⁵ HeLa-cells purified with the 3 μm Hydrolonmembrane;Lane 2: RNA from 5×10⁵ HeLa-cells purified with the Supor-450 PRmembrane;Lane 3: RNA from 5×10⁵ HeLa-cells purified with the 3 μm Fluoroporemembrane;

EXAMPLE 30 Restriction of Plasmid DNA with the Ava I Enzyme on aHydrophobic Membrane

According to the procedures of Example 1, plastic columns were assembledwith hydrophobic membranes (e.g., Supor-200 PR from Pall).

100 μl of a plasmid-containing aqueous solution (pCMVβ by Clontech) weremixed with 350 μl of a lysis buffer containing guanidiniumisothiocyanate (4 M GITC, 0.1 M MgSO₄, 25 mM sodium acetate, pH 4).Subsequently, 250 μl of isopropanol were added and mixed by pipetting.This mixture was then introduced to the column and passed through themembrane, washed and dried according to Example 4.

100 μl of a 1× buffer for the restriction enzyme Ava I were placed onthe membrane and either: (1) removed, transferred to a new reactiondevice and subsequently treated with the restriction enzyme (i.e., Ava Iby Promega); or (2) a restriction enzyme (i.e., Ava I by Promega) wasadded directly to the eluate in the column.

The reaction mixtures were incubated for 1 hour at 37° C. andsubsequently placed on a non-denaturing gel and analyzed (see FIG. 11).

FIG. 11 shows an ethidium-bromide stained agarose gel of anelectrophoretic separation of pCMVβ-plasmid after restriction with Ava I

Lane 1: uncut plasmid;Lanes 2 & 3: elution with the reaction buffer for Ava I, restrictionreaction in a separate device;Lane 4 & 5: restriction with Ava I on the membrane.

EXAMPLE 31 Pressure Filtration for Isopropanol Precipitation of DNA

The isolation of plasmid DNA was performed according to standardprotocols including the elution step via anion exchange chromatography.The DNA was eluted from the column in a high saline buffer.

Subsequently, 0.7 volume of isopropanol was added to this DNA solution,the sample was mixed and incubated for 1-5 minutes at room temperature.A 0.45 μm cellulose acetate filter with a 5 cm² surface in a filtrationcartridge (standard installation for sterile filtration, e.g., Minisartby Sartorius) was used as a filtration installation. This filter wasconnected to a syringe from which the plunger has been removed first.The syringe was then filled with the DNA/isopropanol mixture and pressedthrough the filter with the syringe plunger. A high percentage of theDNA in this precipitate stays on the filter (i.e., cannot pass thepores).

The plunger was again removed from the syringe, was inserted again, andair was pressed through the filter. This step was repeated once or twiceand serves to dry the membrane.

Subsequently, elution was performed with a corresponding volume of a lowsaline buffer, whereby the buffer fills the syringe and was pressedthrough the filter with the plunger. To increase the yield, this firsteluate was again put into the syringe and pressed through the filterwith the plunger. In this test configuration, the yields obtainedtypically range from 80 to 90% (see Example 34).

EXAMPLE 32 Vacuum Filtration for the Isopropanol Precipitation of DNA

As with pressure filtration, first plasmid DNA was isolated and mixedwith 0.7 volume isopropanol. An apparatus designed for vacuum filtrationwas used as a filtration installation, in which a 0.45 μm celluloseacetate filter with a surface of 5 cm² was placed. 0.45 μm cellulosenitrate filters or several layers of 0.65 μm cellulose acetate orcellulose nitrate filters may be used. The isopropanol-DNA mixture wasincubated for 1-5 minutes and placed on the filter assembly. By creatinga vacuum, the solution was suctioned through the filter. TheDNA-precipitates on the filter were mixed with a corresponding volume of70% ethanol and washed by creating a vacuum. The elution of the DNA fromthe filter takes place by adding a low salt buffer, a short incubationand renewed creation of a vacuum. The yield can either be obtained byrepeated elution from the filter with a second volume of low salinebuffer or by elution with the eluate from the first elution step. Herealso, typical yields range from 80%-90% of the DNA.

EXAMPLE 33

The method used is the vacuum filtration method described in Example 32.The filter device used is the vacuum filter apparatus, Sartorius 16315.pCMVβ was used as the plasmid DNA, which was isolated from DH5α cells.

Procedure: In each test, 15 ml of QF-buffer (high saline buffer) aremixed with 500 μg of plasmid. 10.5 ml of isopropanol are added and thisis mixed again. Then the mixture is left to incubate for 5 minutes. Theplasmid DNA thus precipitated is deposited on the membrane in the filterassembly. Next a vacuum is created and the filtration takes place. Themembranes are washed with 5 ml of 70% ethanol (by creating anothervacuum), then 1 ml TE-buffer is pipetted onto the membranes, left toincubate for 5 minutes, and the DNA is eluted by creating a vacuum.Subsequently a post-elution is performed with 1 ml TE-buffer. Total DNAamounts are measured in the flow-through, in the washing stage and inthe combined eluate (OD260). The following results were obtained:

Test Flow- Washing Flow Membrane Number through Stage Eluate Speed PVDF0.2 μm 1 0 μg DNA 0 μg DNA 131 μg Very slow DNA Cellulose Nitrate 2 0 μgDNA 0 μg DNA 418 μg Fast 0.65 μm DNA Cellulose Acetate 3 0 μg DNA 0 μgDNA 469 μg Fast 0.65 μm DNA

Calculated on the basis of 500 μg of DNA starting quantity, thefollowing yields are obtained with this method:

PVDF 0.2 μm 26% Cellulose Acetate 0.65 μm 94% Cellulose Nitrate 0.65 μm84%

EXAMPLE 34

The pressure filtration method indicated in Example 31 was used. Thefilter assembly used was a commercially available 0.45 μm celluloseacetate filter (Minisart, Sartorius). pCMVβ is used as plasmid DNA,which was isolated from DH5α cells.

Procedure: For each test, 15 ml of QF-buffer (high salt buffer) areadded to and mixed with 100, 200, 300, etc., up to 900 μg of plasmid.10.5 ml isopropanol are added and again mixed. Subsequently, there is a5-minute incubation period. The plasmid DNA thus precipitated istransferred to a syringe, to which the filter had been previouslyfitted. Pressure filtration takes place with the aid of the syringe. Thefilter is then washed with 2 ml of 70% ethanol and, as described, driedtwice. The elution is performed with 2 ml of TE-buffer. A second elutionis performed with the eluate. The total amount of DNA is measured in thecombined eluate (OD260).

Following the above procedure, the following results were obtained:

DNA-quantities DNA-quantities used eluted % Yield 100 μg 100 μg 100% 200 μg 176 μg 88% 300 μg 257 μg 86% 400 μg 361 μg 90% 500 μg 466 μg 93%600 μg 579 μg 97% 700 μg 671 μg 96% 800 μg 705 μg 88% 900 μg 866 μg 96%

EXAMPLE 35

The vacuum filtration method indicated in Example 32 was used. Thefilter assembly used was a commercially obtained 0.45 gm celluloseacetate filter (Minisart, Sartorius), that had been attached to afiltration chamber (QIAvac). As buffer reservoir, a syringe was attachedto the other end of the filter. pCMVβ was used as plasmid DNA, which wasisolated from DH5α cells.

Procedure: 15 ml of QF-buffer (high saline buffer) are added to andmixed with 500 μg of plasmid. 10.5 ml isopropanol are added and againmixed. Subsequently, there is a 5-minute incubation period. The plasmidDNA thus precipitated is then transferred to the filter assemblysyringe. Now a vacuum is created and filtration takes place. The filteris not washed with 70% ethanol. Rather, elution with 2 ml of EB buffer(QIAGEN) follows immediately. Post-elution is performed with the eluate.The total DNA quantity in the combined eluate is measured (OD260). Thefollowing result was obtained:

% Test Number Eluted DNA Yield 1 434 μg 87% 2 437 μg 87%

Although a number of embodiments have been described above, it will beunderstood by those skilled in the art that modifications and variationsof the described devices and methods may be made without departing fromconcept of the invention as defined in the appended claims. The articlesand other publications cited herein are incorporated by reference.

1. An isolation device adapted to the isolation of nucleic acidscomprising: at least one cylindrical upper part having an upper openingand a bottom opening; a membrane located at the bottom opening spanningthe entire cross-section of the bottom opening; a bottom part containingan absorbent material; and a mechanism for connecting the upper andbottom parts, such that, after the connection is made, the membrane isin contact with the absorbent material and, in case the connection isnot made, the membrane is not in contact with the absorbent material. 2.The isolation device according to claim 1, wherein the bottom part is acylinder having the same diameter as the upper part.
 3. The isolationdevice according to claim 1, wherein the mechanism for connecting theupper and bottom parts also permits a spatial separation of the upperand bottom parts.
 4. The isolation device according to claim 1, whereinthe connection mechanism is a bayonet socket.
 5. The isolation deviceaccording to claim 1, wherein the connection mechanism is a threadedsocket.
 6. The isolation device according to claim 1, wherein themechanism for connecting the upper and bottom parts includes a slidingmember, which can be slid between the absorbent material and themembrane, to separate the upper and bottom parts.
 7. The isolationdevice according to claim 1, wherein the connection mechanism has apredetermined breaking point between the upper and bottom part.
 8. Theisolation device according claim 1, wherein the upper part is a tube,which is suitable for placement in a reaction container holder.
 9. Theisolation device according to claim 1, wherein the upper and bottomparts form a tube, which is suitable for placement in a reactioncontainer holder.
 10. The isolation device according to claim 1, whereinsaid bottom part is configured to connect with a plurality of upperparts.
 11. The isolation device according to claim 1, wherein saidmembrane is composed of a material selected from the group consisting ofcellulose acetate; non-carboxylized, hydrophobic polyvinylidenefluoride; and massive, hydrophobic polytetrafluoroethylene.
 12. Theisolation device according to claim 11 wherein said membrane material isin the form of a granulate.
 13. The isolation device according to claim11, wherein said membrane material is in the form of a fiber.
 14. Theisolation device according to claim 13, wherein the fibers are organizedas a fleece.
 15. The isolation device according to any one of claims 1to 14, wherein the absorbent material is a sponge.
 16. The isolationdevice according to any one of claims 1 to 14, wherein the absorbentmaterial comprises granules.
 17. A method of using an isolation deviceaccording to claim 1 for isolation of nucleic acids comprising: charginga nucleic acid-containing fluid sample into the upper part of theisolation device of claim 1 so that the sample contacts the uppersurface of said membrane; immobilizing the nucleic acids on saidmembrane; and contacting said absorbent material with said membrane todraw the fluid parts of the sample through the membrane.
 18. The methodof use according to claim 17, further comprising the step of: analyzingat least one property of said nucleic acids in said isolation device.19. The method of use according to claim 17, further comprising the stepof: performing at least one chemical reaction with said nucleic acids insaid isolation device.
 20. The method of use according to claim 19,wherein said at least one chemical reaction is amplification of saidnucleic acids.
 21. An isolation device adapted for the isolation ofnucleic acids comprising: at least one upper part having an upperopening, a bottom opening, and a membrane which is located at the bottomopening of said upper part and which covers the entire cross-section ofsaid bottom opening; a bottom part having an absorbent material; and acollar surrounding the upper part at least in the area of the membranecontaining a coolant.
 22. The isolation device according to claim 21,wherein said collar has two compartments, which are separated from oneanother by a frangible separation wall; and wherein each of thecompartments contains a solution, wherein mixture of the respectivesolutions in the two compartments produces a coolant.
 23. A kit for theisolation of nucleic acids comprising: an immobilization buffer; anelution buffer; and at least one isolation device according to claim 1.24. The kit according to claim 23, further comprising a washing buffer.25. The kit according to claim 23 or 24, further comprising a lysisbuffer.